Substituted 4-methyl-pyrrolo[1,2-a]pyrimidine-8-carboxamide compounds and uses thereof for modulating glucocerebrosidase activity

ABSTRACT

Disclosed are new small molecules having a 4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide core structure and the uses thereof for modulating glucocerebrosidase activity. Also disclosed are pharmaceutical compositions comprising the small molecules which may be administered in methods of treating diseases or disorders associated with glucocerebrosidase activity, including neurological diseases and disorders such as Gaucher&#39;s disease and Parkinson&#39;s disease. The small molecules may contain a fluorophore or may be conjugated to a fluorophore in order to prepare a fluorescent probe for use in high throughput screening methods to identify new modulators of glucocerebrosidase activity via fluorescence polarization.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C.119(e) to U.S. Provisional Patent Application No. 62/187,463, filed onJul. 1, 2015, the content of which is incorporated herein by referencein its entirety.

BACKGROUND

The field of the invention relates to new small molecules and uses ofthe new small molecules for modulating glucocerebrosidase activity. Thenew small molecules have a substituted4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide core structure, such asa 2,4-dimethylpyrrrolo[1,2-a]pyrimidine-8-carboxamide core structure,and the small molecules may be administered to treat diseases anddisorders associated with aberrant glucocerebrosidase activity includingneurodegenerative diseases, such as Gaucher's disease and Parkinson'sdisease.

Glucocerebrosidase (EC 3.2.1.45), which also is calledβ-glucocerebrosidase, β-glucosidase, D-glucosyl-N-acylsphingosineglucohydrolase, or GCase, is an enzyme having glucosylceramidaseactivity. Glucocerebrosidase is required to cleave the beta-glucosidiclinkage of the chemical glucocerebroside, which is an intermediate inglycolipid metabolism. Glucocerebrosidase is localized in the lysosomeand disabling mutations in the gene for glucocerebrosidase (GBA1) areassociated with abnormal accumulation of lipids in lysosomes.

Genetic diseases caused by mutations in GBA1 include neurodegenerativediseases such as Gaucher's disease and Parkinson's disease. Gaucher'sdisease is a rare genetic disease caused by GBA1 gene mutations.Currently, the treatment for Type 1 Gaucher's disease is enzymereplacement therapy (ERT) administered every two weeks. ERT is veryexpensive and not effective for neuronopathic forms of Gaucher'sdisease. Mutations in GBA1 also are linked to Parkinson's disease (PD)by increasing the risk of PD. The so-called “pharmacological chaperonestrategy” has been previously attempted in order to activate GCase.However, none of the compounds used in the pharmacological chaperonestrategy were successful in activating GCase presumably because theytargeted the active site of GCase.

Here, we disclose novel substituted4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide compounds which modulateglucocerebrosidase activity. The substituted4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide compounds disclosedherein have better chemical and physical properties than previousreported non-active site GCase inhibitors. (See Goldin et al., WO,“Substituted pyrazolopyrimidines as glucocerebrosidase activators.”December 2010, WO2012078855; and Patnaik et al., “Discovery,structure-activity relationship, and biological evaluation ofnoninhibitory small molecule chaperones of glucocerbrosidase,” J. Med.Chem. 2012 Jun. 28; 55(1′2):5734-48, the contents of which areincorporated herein by reference in their entireties). These betterchemical and physical properties of the disclosed4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide compounds include polarsurface area, solubility, increased number of rotatable bonds, andincreased number of potential hydrogen bonding members. Some of thesubstituted 4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide compounds inthe present study are capably of highly activating GCase. For example,some of the substituted 4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamidecompounds bind to GCase covalently and activate wild-type GCase up to15-30 fold. GCase thus activated by the novel compounds is observed tobe more stable in an acidic environment than non-activated GCase.Moreover, we found that the substituted4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide compounds can beconjugated to fluorophones to create pyrrolopyrimidine fluorescentprobes which show strong binding affinity in fluorescence polarizationassays. This suggests that these pyrrolopyrimidine fluorescent probesmay be utilized in high throughput screening methods to identify furthermodulators of GCase activity.

SUMMARY

Disclosed are new small molecules having a substituted4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide core structure and usesof the small molecules for modulating glucocerebrosidase activity. Thenew small molecules preferably modulate glucocerebrosidase activity bybinding to glucocerebrosidase, optionally covalently, and activatingglucocerebrosidase. The new small molecules may be formulated aspharmaceutical compositions that comprise the small molecules or thatcomprise activated glucocerebrosidase conjugated to the small molecules,which compositions may be administered in methods of treating and/orpreventing diseases or disorders associated with glucocerebrosidaseactivity, including neurological diseases and disorders such asGaucher's disease and Parkinson's disease. The disclosed small moleculesalso may comprise fluorophores or may be conjugated to fluorophores togenerate fluorescent probes. The fluorescent probes contemplated hereinmay exhibit fluorescence polarization and may be utilized in highthroughput screening methods to identify novel modulators ofglucocerebrosidase.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Structures of GCase activators and probes.

FIG. 2. Dose-response curves for the GCase activators 1, 2 and 4 with A)4MU-Glc substrate; and B) red substrate.

FIG. 3. FP curves for probes 3 and compete assay with compound 1 and 2.

FIG. 4. GCase was activated by probe 4 in a time-dependent manner for A)4MU-β-glc substrate; B) Res-β-glc substrate, and (C) natural substrate.

FIG. 5. The wt. and pre-activated GCase were activated by saposin C in adose-dependent manner.

FIG. 6. Enzyme activity of the activated enzyme and wt. enzyme in pH 4.7buffer and human plasma. A) Enzyme activity of activated GCase vs. wt.GCase tested by 4MU-β-glc substrate enzyme activity assay in pH 4.7buffer; B) Normalization of the enzyme activity in pH 4.7 buffer; C)Enzyme activity of activated GCase vs wt. GCase tested by 4MU-β-glcsubstrate enzyme activity assay in human plasma; D) Normalization of theenzyme activity in human plasma.

FIG. 7. Cell uptake assay (U937 macrophage). A) Enzyme activity ofactivated GCase vs wt. GCase tested by 4MU-β-glc substrate enzymeactivity assay; B) Western blot: endogenous, activated and wt. GCase.

DETAILED DESCRIPTION

The disclosed subject matter further may be described utilizing terms asdefined below.

Unless otherwise specified or indicated by context, the terms “a”, “an”,and “the” mean “one or more.” For example, “a modulator ofglucocerebrosidase activity” should be interpreted to mean “one or moremodulators of glucocerebrosidase activity.”

As used herein, “about”, “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” and“approximately” will mean plus or minus ≦10% of the particular term and“substantially” and “significantly” will mean plus or minus ≧10% of theparticular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.” The terms “comprise”and “comprising” should be interpreted as being “open” transitionalterms that permit the inclusion of additional components further tothose components recited in the claims. The terms “consist” and“consisting of” should be interpreted as being “closed” transitionalterms that do not permit the inclusion additional components other thanthe components recited in the claims. The term “consisting essentiallyof” should be interpreted to be partially closed and allowing theinclusion only of additional components that do not fundamentally alterthe nature of the claimed subject matter.

The terms “subject,” “patient,” and “individual” may be usedinterchangeably herein. A subject may be a human subject. A subject mayrefer to a human subject having or at risk for acquiring a disease ordisorder that is associated with aberrant glucocerebrosidase activity.As used herein, the term “aberrant” means higher or lower activityrelative to a normal healthy subject. In specific embodiments, a subjectexhibiting aberrant glucocerebrosidase have or be at risk for acquiringa neurological disease or disorder, including degenerative neurologicaldiseases or disorders such as Gaucher's disease and Parkinson's diseaseassociated with aberrant glucocerebrosidase activity.

As used herein, the phrase “effective amount” shall mean that drugdosage that provides the specific pharmacological response for which thedrug is administered in a significant number of patients in need of suchtreatment. An effective amount of a drug that is administered to aparticular patient in a particular instance will not always be effectivein treating the conditions/diseases described herein, even though suchdosage is deemed to be a therapeutically effective amount by those ofskill in the art.

The term “alkyl” as contemplated herein includes a straight-chain orbranched alkyl radical in all of its isomeric forms. Similarly, the term“alkoxy” refers to any alkyl radical which is attached via an oxygenatom (i.e., a radical represented as “alkyl-O—*”). As used herein, anasterick “*” is used to designate the point of attachment for anyradical group or substituent group.

As used herein, the term “modulate” means decreasing or inhibitingactivity and/or increasing or augmenting activity. For example,modulating glucocerebrosidase activity means decreasing or inhibitingglucocerebrosidase activity and/or increasing or augmentingglucocerebrosidase activity. The compounds disclosed herein may beadministered to modulate glucocerebrosidase activity for example, as achaperone or activator.

The compounds disclosed herein may be referred to as“4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide compounds.” Thecompounds or salt or solvates thereof may be described as having aFormula I as follows:

wherein:

R¹ is hydrogen; a C1-C6 alkyl group; a C2-C6 alkenyl group; a C2-C6alkynyl group; a saturated or unsaturated homocycle or heterocyclecomprising one 5- or 6-membered ring; a saturated or unsaturatedhomocycle or heterocycle comprising two fused 5- or 6-membered rings, oran alkylthiophene (e.g., 2-methylthiophene); and R¹ optionally issubstituted at one or more positions with a C1-C6 alkyl group; a C1-C6alkoxy group; a halo group; a haloalkyl group; phenyl group (whichoptionally is substituted with halo); a benzyl group (which optionallyis substituted with halo); a triazole group (optionally substituted witha carboxyl group); a 2,5-dioxopyrrolidinyl-1-yl-carboxylate group; anamino group; an alkyl-N,N-dialkyl amino group; an alkyl-alkyoxy-aminogroup; an alkyl-alkyoxy-alkoxy-amino group; analkyl-alkyoxy-alkoxy-carboxamide-alkyl-morpholine group; analkyl-alkyoxy-alkoxy-carboxamide-alkyl-1-alkylpyrrolidine group; analkyl-alkyoxy-alkoxy-carboxamide-alkyl-cyclohexyl group; or analkyl-alkyoxy-alkoxy-carboxamide-alkyl-cyclobutyl group); an imidazolegroup; a pyridyl group (e.g., 2-yl, 3-yl, or 4-yl, optionallysubstituted with phenoxy); a pyrrolidinyl group; a piperazinyl group; a4-alkylpiperazine group; a 4-benzylpiperazine group; analkyl-4-alkylpiperazine group; a piperidinyl group; a 4-alkylpiperidinegroup; a 4-N,N,diakylaminopiperidine group; a morpholinyl group; analkylmorpholine group; an amino group; an alkylamino group; a dialkylamino group; an alkyl-N,N, dialkylamino group; an azide group; ahydroxyl group; an alkylhydroxyl group; an alkynylphenyl group; aphenylmethanone group; an oxyphenyl group; an oxycarboxyl group; or R′has a formula selected from:

and R³ has a formula selected from

and R⁴ is H, C1-C8 alkyl, phenyl, or succinimidyl (e.g.,N-succinimidyl); and

R² is C1-C6 alkyl or pyridinyl (e.g., 2-yl, 3-yl, or 4-yl). Compounds inwhich R² is methyl in particular are disclosed and may be referred to as2,4-dimethylpyrrrolo[1,2-a]pyrimidine-8-carboxamide compounds. Compoundsin which R² is 3-yl-pyridine in particular are disclosed and may bereferred to as2-(pyridin-3-yl)-4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamidecompounds.

In some embodiments, R¹ is selected from the group consisting of

where the asterick (*) designates the point of attachment of R¹ to the8-carboximide nitrogen atom.

In some embodiments, the disclosed compounds have a Formula IA:

wherein:

R⁵ is hydrogen a C1-C6 alkyl group; a C2-C6 alkenyl group; a C2-C6alkynyl group; a C1-C6 alkoxy group; a halo group; a haloalkyl group; aphenyl group; a benzyl group; a triazole group (optionally substitutedwith a carboxyl group, a 2,5-dioxopyrrolidinyl-1-yl-carboxylate group,an amino group, an alkyl-N,N-dialkyl amino group, an alkyl-alkyoxy-aminogroup, an alkyl-alkyoxy-alkoxy-amino group, analkyl-alkyoxy-alkoxy-carboxamide-alkyl-morpholine group, analkyl-alkyoxy-alkoxy-carboxamide-alkyl-1-alkylpyrrolidine group, analkyl-alkyoxy-alkoxy-carboxamide-alkyl-cyclohexyl group, or analkyl-alkyoxy-alkoxy-carboxamide-alkyl-cyclobutyl group); an imidazolegroup; a pyrrolidine group; a piperazine group; a 4-alkylpiperazinegroup; a 4-benzylpiperazine group; an alkyl-4-alkylpiperazine group; apiperidine group; a 4-alkylpiperidine group; a4-N,N,diakylaminopiperidine group; a morpholine group; analkylmorpholine group; an amino group; an alkylamino group; a dialkylamino group; an alkyl-N,N-dialkylamino group; an azide group; a hydroxylgroup; an alkylhydroxyl group; an alkynylphenyl group; a phenylmethanonegroup; an oxyphenyl group; or an oxycarboxyl acid group.

In some embodiments, the disclosed compounds have a Formula IB:

wherein:

R is a carboxyl group, a 2,5-dioxopyrrolidinyl-1-yl-carboxylate group;an alkylamino group; an alkyl-N,N-dialkyl amino group; analkyl-alkyoxy-amino group; an alkyl-alkyoxy-alkoxy-amino group; analkyl-alkyoxy-alkoxy-carboxamide-alkyl-morpholine group; analkyl-alkyoxy-alkoxy-carboxamide-alkyl-1-alkylpyrrolidine group; analkyl-alkyoxy-alkoxy-carboxamide-alkyl-cyclohexyl group; and analkyl-alkyoxy-alkoxy-carboxamide-alkyl-cyclobutyl group.

The disclosed compounds may comprise or may be conjugated to afluorophore. In some embodiments of the disclosed compounds, any ofsubstituents R¹, R₂, R₃, R₄, R₅, and R⁶, may comprise a fluorophone,including fluorophores suitable for use in fluorescence polarizationassays. As used herein, a “fluorophore” is a chemical group that can beexcited (e.g., by light or a chemical reaction) to emit fluorescence.Some suitable fluorophores may be excited by light to emitphosphorescence. As used herein, a “dye” may include a fluorophore. Thedithio compounds described herein may include fluorophore selected frombut not limited to: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein);5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX(carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine);6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin;7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin;9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA(9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red;Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Alexa Fluor 350;Alexa Fluor 430; Alexa Fluor 488; Alexa Fluor 532; Alexa Fluor 546;Alexa Fluor 568; Alexa Fluor 594; Alexa Fluor 633; Alexa Fluor 647;Alexa Fluor 660; Alexa Fluor 680; Alizarin Complexon; Alizarin Red;Allophycocyanin (APC); AMC; AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X;Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); AnilinBlue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTS; AstrazonBrilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G;Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); Berberine Sulphate;Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein;BFP/GFP FRET; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); BlancophorFFG; Blancophor SV; BOBO™-1; BOBO™-3; Bodipy 492/515; Bodipy 493/503;Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy FL; Bodipy FL ATP;Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate;Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1;BO-PRO™-3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; CalciumCrimson™; Calcium Green; Calcium Orange; Calcofluor White;Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow;Catecholamine; CCF2 (GeneBlazer); CFDA; CFP-Cyan Fluorescent Protein;CFP/YFP FRET; Chlorophyll; Chromomycin A; CL-NERF (Ratio Dye, pH);CMFDA; Coelenterazine f; Coelenterazine fcp; Coelenterazine h;Coelenterazine hcp; Coelenterazine ip; Coelenterazine n; CoelenterazineO; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTCFormazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; CyanGFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine;Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI;Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (DichlorodihydrofluoresceinDiacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS(non-ratio); DiA (4-Di-16-ASP); Dichlorodihydrofluorescein Diacetate(DCFH); DiD-Lipophilic Tracer; DiD (DiICl8(5)); DIDS; Dihydorhodamine123 (DHR); DiI (DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR(DiIC18(7)); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP;ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide;Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III)chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FITC; FlazoOrange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate;Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX;FM 1-43; FM 4-46; Fura Red™; Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF;Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP (S65T); GFP red shifted(rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, UVexcitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue;Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS;Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine;Indo-1; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); IntrawhiteCf; JC-1; JO-JO-1; JO-PRO-1; Laurodan; LDS 751 (DNA); LDS 751 (RNA);Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine;Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1;Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso TrackerGreen; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue;LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red(Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; MagnesiumGreen; Magnesium Orange; Malachite Green; Marina Blue; Maxilon BrilliantFlavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin;Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; MitotrackerRed; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH);Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine;Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; NuclearYellow; Nylosan Brilliant Iavin EBG; Oregon Green; Oregon Green 488-X;Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514;Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP;PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); PhorwiteAR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613[PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110;Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green;Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; RhodamineWT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); RsGFP; S65A;S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange;Sevron Yellow L; sgBFP™; sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (superglow GFP); SITS; SITS (Primuline); SITS (Stilbene IsothiosulphonicAcid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; SodiumGreen; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ(6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange;Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange;Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange;Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5;TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITCTetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; UranineB; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F;Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3. Asused herein, a “fluorophore” may include a salt of the fluorophore.

In some embodiments, the disclosed compounds have a Formula ID:

wherein:

X is N or S; and

R⁴, R⁵, R⁶, and R⁷, are each independently selected from hydrogen, C1-C6alkyl, C1-C6 alkoxy, and halogen.

The disclosed compounds may include two substituted4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide groups conjugated via alinker. Compounds that include two substituted4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide groups conjugated via alinker between the carboxamide groups may be illustrated as follows:

In some embodiments, the disclosed compounds having two substituted4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide groups conjugated via alinker may be described as having a Formula II as follows:

In some embodiments, the disclosed compounds having two substituted4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide groups conjugated via alinker may be described as having a Formula III as follows:

In some embodiments, the disclosed compounds having two substituted4-methylpyrrrolo[1,2-a]pyrimidine-8-carboxamide groups conjugated via alinker may be described as having a Formula IV as follows:

The compounds disclosed herein preferably modulate activity ofglucocerebrosidase. Modulation may include inhibiting or decreasingglucocerebrosidase activity. Modulation also may include activating orincreasing glucocerebrosidase activity. Glucocerebrosidase activity maybe assessed utilizing methods known in the art and the methods disclosedherein, including the methods disclosed in the Examples provided herein.In some embodiments, the compounds decrease or increaseglucocerebrosidase activity relative to a control (e.g., by at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more). In otherembodiments, an AC₅₀ value or IC₅₀ value for the compound in regard toinhibition or activation of glucocerebrosidase may be determined andpreferably the compound has an AC₅₀ value or IC₅₀ value of less thanabout 10 μM, 5 μM, or 1 μM, 0.5 μM, 0.1 μM, 0.05 μM, 0.01 μM, 0.005 μM,or 0.001 μM.

The compounds disclosed herein (e.g., compounds of Formula I) may haveseveral chiral centers, and stereoisomers, epimers, and enantiomers arecontemplated. The compounds may be optically pure with respect to one ormore chiral centers (e.g., some or all of the chiral centers may becompletely in the S configuration; some or all of the chiral centers maybe completely in the R configuration; etc.). Additionally oralternatively, one or more of the chiral centers may be present as amixture of configurations (e.g., a racemic or another mixture of the Rconfiguration and the S configuration). Compositions comprisingsubstantially purified stereoisomers, epimers, or enantiomers, oranalogs or derivatives thereof are contemplated herein (e.g., acomposition comprising at least about 90%, 95%, or 99% purestereoisomer, epimer, or enantiomer.) As used herein, formulae which donot specify the orientation at one or more chiral centers are meant toencompass all orientations and mixtures thereof.

The compounds employed in the compositions and methods disclosed hereinmay be administered as pharmaceutical compositions and, therefore,pharmaceutical compositions incorporating the compounds are consideredto be embodiments of the compositions disclosed herein. Suchcompositions may take any physical form which is pharmaceuticallyacceptable; illustratively, they can be orally administeredpharmaceutical compositions. Such pharmaceutical compositions contain aneffective amount of a disclosed compound, which effective amount isrelated to the daily dose of the compound to be administered. Eachdosage unit may contain the daily dose of a given compound or eachdosage unit may contain a fraction of the daily dose, such as one-halfor one-third of the dose. The amount of each compound to be contained ineach dosage unit can depend, in part, on the identity of the particularcompound chosen for the therapy and other factors, such as theindication for which it is given. The pharmaceutical compositionsdisclosed herein may be formulated so as to provide quick, sustained, ordelayed release of the active ingredient after administration to thepatient by employing well known procedures.

The compounds for use according to the methods of disclosed herein maybe administered as a single compound or a combination of compounds. Forexample, a compound that modulates glucocerebrosidase activity may beadministered as a single compound or in combination with anothercompound that modulates glucocerebrosidase activity or that has adifferent pharmacological activity.

As indicated above, pharmaceutically acceptable salts of the compoundsare contemplated and also may be utilized in the disclosed methods. Theterm “pharmaceutically acceptable salt” as used herein, refers to saltsof the compounds which are substantially non-toxic to living organisms.Typical pharmaceutically acceptable salts include those salts preparedby reaction of the compounds as disclosed herein with a pharmaceuticallyacceptable mineral or organic acid or an organic or inorganic base. Suchsalts are known as acid addition and base addition salts. It will beappreciated by the skilled reader that most or all of the compounds asdisclosed herein are capable of forming salts and that the salt forms ofpharmaceuticals are commonly used, often because they are more readilycrystallized and purified than are the free acids or bases.

Acids commonly employed to form acid addition salts may includeinorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, phosphoric acid, and the like, and organic acidssuch as p-toluenesulfonic, methanesulfonic acid, oxalic acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of suitablepharmaceutically acceptable salts may include the sulfate, pyrosulfate,bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide,acetate, propionate, decanoate, caprylate, acrylate, formate,hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate,propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate,maleat-, butyne-.1,4-dioate, hexyne-1,6-dioate, benzoate,chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate,phthalate, xylenesulfonate, phenylacetate, phenylpropionate,phenylbutyrate, citrate, lactate, alpha-hydroxybutyrate, glycolate,tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate, and the like.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Bases useful in preparing such salts includesodium hydroxide, potassium hydroxide, ammonium hydroxide, potassiumcarbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate,calcium hydroxide, calcium carbonate, and the like.

The particular counter-ion forming a part of any salt of a compounddisclosed herein is may not be critical to the activity of the compound,so long as the salt as a whole is pharmacologically acceptable and aslong as the counterion does not contribute undesired qualities to thesalt as a whole. Undesired qualities may include undesirably solubilityor toxicity.

Pharmaceutically acceptable esters and amides of the compounds can alsobe employed in the compositions and methods disclosed herein. Examplesof suitable esters include alkyl, aryl, and aralkyl esters, such asmethyl esters, ethyl esters, propyl esters, dodecyl esters, benzylesters, and the like. Examples of suitable amides include unsubstitutedamides, monosubstituted amides, and disubstituted amides, such as methylamide, dimethyl amide, methyl ethyl amide, and the like.

In addition, the methods disclosed herein may be practiced using solvateforms of the compounds or salts, esters, and/or amides, thereof. Solvateforms may include ethanol solvates, hydrates, and the like.

The pharmaceutical compositions may be utilized in methods of treating adisease or disorder associated glucocerebrosidase activity. For example,the pharmaceutical compositions may be utilized to treat patients havingor at risk for acquiring a neurological disease or disorder, includingdegenerative neurological diseases or disorders such as Gaucher'sdisease and Parkinson's disease. Suitable patients include, for examplemammals, such as humans and non-human primates (e.g., chimps) or othermammals (e g., dogs, cats, horses, rats, and mice). Suitable humanpatients may include, for example, those who have previously beendetermined to be at risk of having or developing a neurological diseaseor disorder, including degenerative neurological diseases or disorderssuch as Gaucher's disease and Parkinson's disease.

As used herein, the terms “treating” or “to treat” each mean toalleviate symptoms, eliminate the causation of resultant symptoms eitheron a temporary or permanent basis, and/or to prevent or slow theappearance or to reverse the progression or severity of resultantsymptoms of the named disease or disorder. As such, the methodsdisclosed herein encompass both therapeutic and prophylacticadministration.

As used herein the term “effective amount” refers to the amount or doseof the compound, upon single or multiple dose administration to thesubject, which provides the desired effect in the subject underdiagnosis or treatment. The disclosed methods may include administeringan effective amount of the disclosed compounds (e.g., as present in apharmaceutical composition) for treating a disease or disorderassociated with superoxide dismutase mutations, including administeringan effective amount of a compound that inhibits expression of themutated form of superoxide dismutase.

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount or dose of compound administered, anumber of factors can be considered by the attending diagnostician, suchas: the species of the subject; its size, age, and general health; thedegree of involvement or the severity of the disease or disorderinvolved; the response of the individual subject; the particularcompound administered; the mode of administration; the bioavailabilitycharacteristics of the preparation administered; the dose regimenselected; the use of concomitant medication; and other relevantcircumstances.

A typical daily dose may contain from about 0.01 mg/kg to about 100mg/kg (such as from about 0.05 mg/kg to about 50 mg/kg and/or from about0.1 mg/kg to about 25 mg/kg) of each compound used in the present methodof treatment.

Compositions can be formulated in a unit dosage form, each dosagecontaining from about 1 to about 500 mg of each compound individually orin a single unit dosage form, such as from about 5 to about 300 mg, fromabout 10 to about 100 mg, and/or about 25 mg. The term “unit dosageform” refers to a physically discrete unit suitable as unitary dosagesfor a patient, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical carrier, diluent, orexcipient.

Oral administration is an illustrative route of administering thecompounds employed in the compositions and methods disclosed herein.Other illustrative routes of administration include transdermal,percutaneous, intravenous, intramuscular, intranasal, buccal,intrathecal, intracerebral, or intrarectal routes. The route ofadministration may be varied in any way, limited by the physicalproperties of the compounds being employed and the convenience of thesubject and the caregiver.

As one skilled in the art will appreciate, suitable formulations includethose that are suitable for more than one route of administration. Forexample, the formulation can be one that is suitable for bothintrathecal and intracerebral administration. Alternatively, suitableformulations include those that are suitable for only one route ofadministration as well as those that are suitable for one or more routesof administration, but not suitable for one or more other routes ofadministration. For example, the formulation can be one that is suitablefor oral, transdermal, percutaneous, intravenous, intramuscular,intranasal, buccal, and/or intrathecal administration but not suitablefor intracerebral administration.

The inert ingredients and manner of formulation of the pharmaceuticalcompositions are conventional. The usual methods of formulation used inpharmaceutical science may be used here. All of the usual types ofcompositions may be used, including tablets, chewable tablets, capsules,solutions, parenteral solutions, intranasal sprays or powders, troches,suppositories, transdermal patches, and suspensions. In general,compositions contain from about 0.5% to about 50% of the compound intotal, depending on the desired doses and the type of composition to beused. The amount of the compound, however, is best defined as the“effective amount”, that is, the amount of the compound which providesthe desired dose to the patient in need of such treatment. The activityof the compounds employed in the compositions and methods disclosedherein are not believed to depend greatly on the nature of thecomposition, and, therefore, the compositions can be chosen andformulated primarily or solely for convenience and economy.

Capsules are prepared by mixing the compound with a suitable diluent andfilling the proper amount of the mixture in capsules. The usual diluentsinclude inert powdered substances (such as starches), powdered cellulose(especially crystalline and microcrystalline cellulose), sugars (such asfructose, mannitol and sucrose), grain flours, and similar ediblepowders.

Tablets are prepared by direct compression, by wet granulation, or bydry granulation. Their formulations usually incorporate diluents,binders, lubricants, and disintegrators (in addition to the compounds).Typical diluents include, for example, various types of starch, lactose,mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such assodium chloride), and powdered sugar. Powdered cellulose derivatives canalso be used. Typical tablet binders include substances such as starch,gelatin, and sugars (e.g., lactose, fructose, glucose, and the like).Natural and synthetic gums can also be used, including acacia,alginates, methylcellulose, polyvinylpyrrolidine, and the like.Polyethylene glycol, ethylcellulose, and waxes can also serve asbinders.

Tablets can be coated with sugar, e.g., as a flavor enhancer andsealant. The compounds also may be formulated as chewable tablets, byusing large amounts of pleasant-tasting substances, such as mannitol, inthe formulation. Instantly dissolving tablet-like formulations can alsobe employed, for example, to assure that the patient consumes the dosageform and to avoid the difficulty that some patients experience inswallowing solid objects.

A lubricant can be used in the tablet formulation to prevent the tabletand punches from sticking in the die. The lubricant can be chosen fromsuch slippery solids as talc, magnesium and calcium stearate, stearicacid, and hydrogenated vegetable oils.

Tablets can also contain disintegrators. Disintegrators are substancesthat swell when wetted to break up the tablet and release the compound.They include starches, clays, celluloses, algins, and gums. As furtherillustration, corn and potato starches, methylcellulose, agar,bentonite, wood cellulose, powdered natural sponge, cation-exchangeresins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, andcarboxymethylcellulose can be used.

Compositions can be formulated as enteric formulations, for example, toprotect the active ingredient from the strongly acid contents of thestomach. Such formulations can be created by coating a solid dosage formwith a film of a polymer which is insoluble in acid environments andsoluble in basic environments. Illustrative films include celluloseacetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, and hydroxypropyl methylcellulose acetatesuccinate.

When it is desired to administer the compound as a suppository,conventional bases can be used. Illustratively, cocoa butter is atraditional suppository base. The cocoa butter can be modified byaddition of waxes to raise its melting point slightly. Water-misciblesuppository bases, such as polyethylene glycols of various molecularweights, can also be used in suppository formulations.

Transdermal patches can also be used to deliver the compounds.Transdermal patches can include a resinous composition in which thecompound will dissolve or partially dissolve; and a film which protectsthe composition and which holds the resinous composition in contact withthe skin. Other, more complicated patch compositions can also be used,such as those having a membrane pierced with a plurality of poresthrough which the drugs are pumped by osmotic action.

As one skilled in the art will also appreciate, the formulation can beprepared with materials (e.g., actives excipients, carriers (such ascyclodextrins), diluents, etc.) having properties (e.g., purity) thatrender the formulation suitable for administration to humans.Alternatively, the formulation can be prepared with materials havingpurity and/or other properties that render the formulation suitable foradministration to non-human subjects, but not suitable foradministration to humans.

The compounds disclosed in the present application may function asactivators of glucocerebrosidase. For example, a compound disclosedherein may be reacted with glucocerebrosidase to prepare an activatedglucocerebrosidase that is covalent attached to the compound. Theactivated glucocerebrosidase thusly formed may be prepared as apharmaceutical composition to treat and/or prevent a disease or disorderthat is associated with glucocerebrosidase activity as in enzymereplacement therapy, which is known in the art.

The following list of formulations is illustrative. These illustrativeformulations may be suitable for preparing pharmaceutical compositionsthat include the disclosed compounds as “active ingredients.” Thefollowing list of formulations is illustrative and should not beinterpreted as limiting the present disclosure or claims in any way:

Formulation 1

Hard gelatin capsules are prepared using the following ingredients:

Quantity (mg/capsule) Active Ingredient 250 Starch, dried 200 Magnesiumstearate 10 Total 460 mg

The above ingredients are mixed and filled into hard gelatin capsules in460 mg quantities.

Formulation 2

Quantity (mg/tablet) Active Ingredient 250 Cellulose, microcrystalline400 Silicon dioxide, fumed 10 Stearic acid 5 Total 665 mg

The components are blended and compressed to form tablets each weighing665 mg.

Formulation 3

An aerosol solution is prepared containing the following components:

Weight % Active Ingredient 0.25 Ethanol 29.75 Propellant 22(chlorodifluoromethane) 70.00 Total 100.00

The active compound is mixed with ethanol and the mixture added to aportion of the Propellant 22, cooled to −30° C. and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remainder of the propellant. The valveunits are then fitted to the container.

Formulation 4

Tablets each containing 60 mg of active ingredient are made as follows:

Active Ingredient 60 mg Starch 45 mg Microcrystalline cellulose 35 mgPolyvinylpyrrolidone  4 mg Sodium carboxymethyl starch 4.5 mg  Magnesiumstearate 0.5 mg  Talc  1 mg Total 150 mg 

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50° C. and passed through a No. 18 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 60 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 150 mg.

Formulation 5

Capsules, each containing 80 mg medicament, are made as follows:

Active Ingredient 80 mg Starch 59 mg Microcrystalline cellulose 59 mgMagnesium stearate  2 mg Total 200 mg 

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 45 sieve, and filled into hard gelatincapsules in 200 mg quantities.

Formulation 6

Suppositories each containing 225 mg of active ingredient may be made asfollows:

Active Ingredient   225 mg Saturated fatty acid glycerides 2,000 mgTotal 2,225 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

Formulation 7

Suspensions each containing 50 mg of medicament per 5 ml dose are madeas follows:

Active Ingredient 50 mg Sodium carboxymethyl cellulose 50 mg Syrup 1.25ml Benzoic acid solution 0.10 ml Flavor q.v. Color q.v. Purified waterto total 5 ml

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl, cellulose and syrup to form a smooth paste.The benzoic acid solution, flavor, and color are diluted with some ofthe water and added with stirring. Sufficient water is then added toproduce the required volume.

Formulation 8

An intravenous formulation containing 100 mg of medicament per 5 ml dosecan be prepared as follows:

Active Ingredient 100 mg Mannitol 100 mg 5N Sodium hydroxide 200 mlPurified water to total  5 ml

EXAMPLES

The followings Examples are illustrative only and are not intended tolimit the scope of the claimed subject matter.

Pyrrolopyrimidine Compounds as Glucocerebrosidase Modulators and TheirApplications

The compounds were prepared using Scheme I below:

Example 1 Preparation ofN-(4-ethynylphenyl)-2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamide(1)

a. Preparation of ethyl 2-amino-1H-pyrrole-3-carboxylate. To a solutionof carbethoxyacetamidine (390 mg, 3.0 mmol) in dried ethyl acetate (20mL) under an argon atmosphere was rapidly added anhydrouschloroacetaldehyde under vigorous stirring at 22° C. The reaction wasstirred for 10 min and heated at reflux for 20 min. The mixture wascooled to room temperature and filtered through silica gel (15 g). Theresidue in the reaction flask was extracted with ethyl acetate (20mL×5), and filtered. The filtrate was collected and evaporated underreduced pressure to give 120 mg (47%) as a light yellow solid.

¹H NMR: (500 MHz, CDCl₃) δ(ppm): 7.92 (br, 1H), 6.28 (t, J=2.8 Hz, 1H),6.15 (dd, J=2.0 Hz, 1), 4.94 (br, 2H), 4.24 (q, J=7.1 Hz, 2H), 1.33 (t,J=7.1 Hz, 3H). ¹³C NMR: (125 MHz, CDCl₃) δ(ppm)): 166.5, 145.4, 110.3,107.5, 94.5, 59.3, 14.7.

b. Preparation of ethyl2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxylate. Under a nitrogenatmosphere, pentane-2,4-dione (14.3 mL, 139 mmol) and ethyl2-amino-1H-pyrazole-3-carboxylate (19.5 g, 127 mmol) were heated in asealed tube with acetic acid (200 mL) at 110° C. overnight. The aceticacid was evaporated under reduced pressure to give the crude product,which was purified by flash chromatography to give 10.0 g (36%) as alight yellow powder.

¹H NMR: (500 MHz, CDCl₃) δ(ppm): 7.39 (d, J=3.4 Hz, 1H), 7.01 (d, J=3.4Hz, 1H), 6.53 (s, 1H), 4.40 (q, J=7.1 Hz, 1H), 2.62 (s, 1H), 2.56 (s,1H), 1.41 (ts, J=7.1 Hz, 1H). ¹³C NMR: (125 MHz, CDCl₃) δ(ppm): 164.3,157.6, 142.3, 141.8, 118.0, 109.0, 106.6, 102.3, 59.9, 29.8, 25.2, 18.3,14.7. ESI-MS m/z: 241 (M+Na⁺), 219 (M+H⁺).

c. Preparation of 2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxylicacid. Ethyl 2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxylate (10.0 g,45.9 mmol) was suspended in EtOH (200 mL) and treated with 3.4 M sodiumhydroxide (80 mL, 275 mmol). The mixture was heated to 90° C. and thenstirred for 1 h. The mixture was cooled to room temperature andneutralized with acetic acid (15.7 mL, 275 mmol) to pH 6-7. The slurrywas filtered, and the solid residue was washed with water and dried invacuum to obtain a yellow powder (7.0 g, 80%).

¹NMR: (500 MHz, d6-DMSO) δ(ppm)): 11.67 (s, 1H), 7.40 (d, J=3.3 Hz, 1H),7.26 (d, J=3.3 Hz, 1H), 6.85 (s, 1H), 2.61 (s, 3H), 2.51 (s, 3H). ¹³CNMR: (125 MHz, d₆-DMSO) δ(ppm)): 164.1, 156.8, 143.6, 141.3, 117.0,108.8, 108.5, 101.0, 24.1, 17.6. ESI-MS m/z: 213 (M+Na⁺), 191 (M+H⁺).

d. Preparation ofN-(4-ethynylphenyl)-2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamide(1). 2,4-Dimethylpyrrolo[1,2-a]pyrimidine-8-carboxylic acid (1.0 g, 5.2mmol), 4-ethynylaniline (0.61 g, 5.2 mmol), and HATU (2.0 g, 5.2 mmol)were dissolved in DMF (10 mL) and then treated withdiisopropylethylamine (2.75 mL, 15.8 mmol). The mixture was stirred at60° C. overnight. The mixture was diluted with water and filtered. Theresidue was washed with water (×2) and dried in vacuum to obtain ayellow solid (1.0 g, 66%).

¹H NMR: (500 MHz, CDCl₃) δ(ppm)): 10.87 (s, 1H), 7.74 (d, J=8.6 Hz, 2H),7.56 (d, J=3.3 Hz, 1H), 7.48 (d, J=8.6 Hz, 2H), 7.07 (d, J=3.3 Hz, 1H),6.53 (s, 1H), 3.04 (s, 1H), 2.63 (s, 3H), 2.58 (s, 3H). ¹³C NMR: (125MHz, CDCl₃) δ(ppm)): 162.5, 155.7, 142.7, 140.3, 139.6, 133.0, 119.0,117.3, 116.0, 108.5, 107.2, 105.6, 84.1, 76.2, 24.8, 18.3. ESI-MS m/z:312 (M+Na⁺).

Example 2 Preparation ofN-(4-(1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)phenyl)-2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamide(2)

A mixture ofN-(4-ethynylphenyl)-2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamide(145 mg, 0.5 mmol), 2-(2-(2-azidoethoxy)ethoxy)ethanamine (87 mg, 0.5mmol) and copper (I) iodide (20 mg) in chloroform (5 mL) was stirred atroom temperature for 3 h. The mixture was filtered, and the filtrate wasconcentrated and purified by flash chromatography to give a yellow foam(130 mg, 56%). The product is a mixture of 1,4-disubstituted and1,5-disubstituted regioisomers (4:1).

The data of the 1,4-disubstituted regioisomers is reported here. ¹H NMR:(500 MHz, CDCl₃) δ(ppm)): 10.82 (s, 1H), 7.92 (s, 1H), 7.83 (d, J=8.6Hz, 2H), 7.80 (d, J=8.6 Hz, 2H), 7.55 (d, J=3.2 Hz, 1H), 7.05 (d, J=3.2Hz, 1H), 6.51 (s, 1H), 4.57 (t, J=5.0 Hz, 2H), 3.90 (t, J=5.1 Hz, 2H),3.63-3.54 (m, 4H), 3.45 (t, J=5.2 Hz, 2H), 2.82 (t, J=5.2 Hz, 2H), 2.63(s, 3H), 2.56 (s, 3H), 2.15 (br, 2H). ¹³C NMR: (125 MHz, CDCl₃) δ(ppm)):162.5, 155.6, 147.8, 142.6, 139.6, 139.5, 128.2, 126.4, 125.5, 120.4,119.6, 119.3, 117.3, 108.4, 107.1, 105.9, 73.5, 70.8, 70.3, 69.7, 50.4,41.8, 24.8, 18.3. ESI-MS m/z: 464 (M+H⁺).

Example 3 Preparation of2-(3,6-Bis(dimethylamino)xanthylium-9-yl)-5-((2-(2-(2-(4-(4-(2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamido)phenyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)carbamoyl)benzoate(3)

N-(4-(1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)phenyl)-2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamide(88 mg, 0.19 mmol), 3,6-bis(dimethylamino)xanthylium-9-yl) (82 mg, 0.19mmol), and HATU (73 mg, 0.19 mmol) were dissolved in DMF (3 mL) and thentreated with diisopropylethylamine (100 μL, 0.57 mmol). The mixture wasstirred at room temperature overnight. The mixture was diluted withwater and filtered. The residue was washed with water (×2), and dried.The solid obtained was purified by flash chromatography to give adark-red solid (45 mg, 27%).

¹H NMR: (500 MHz, CDCl₃) δ(ppm)): 10.84 (s, 1H), 8.35 (s, 1H), 8.19 (dd,J=8.0, 1.2 Hz, 1H), 7.92 (s, 1H), 7.84 (d, J=8.6 Hz, 2H), 7.79 (d, J=8.6Hz, 2H), 7.57 (d, J=3.3 Hz, 1H), 7.24 (d, J=8.0 Hz, 1H), 7.10 (t, J=4.9Hz, 1H), 7.07 (d, J=3.3 Hz, 1H), 6.55 (d, J=8.0 Hz, 2H), 6.53 (s, 1H),6.46 (d, J=2.4 Hz, 2H), 6.35 (dd, J=8.9, 2.5 Hz, 2H), 4.64 (t, J=5.1 Hz,2H), 4.02 (t, J=5.1 Hz, 2H), 3.71-3.61 (m, 8H), 2.96 (s, 12H), 2.64 (s,3H), 2.59 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃) δ(ppm)): 169.2, 166.2,162.5, 155.7, 153.1, 152.3, 147.8, 142.6, 139.7, 139.5, 136.3, 134.4,128.8, 128.2, 126.4, 125.3, 124.8, 123.0, 120.5, 119.7, 117.3, 108.9,108.5, 107.1, 106.3, 105.8, 98.5, 70.6, 70.5, 69.6, 50.4, 40.3, 40.1,24.8, 18.3. ESI-MS m/z: 876 (M+H⁺), 898 (M+Na⁺).

Example 4 Preparation of 2,5-Dioxopyrrolidin-1-yl3-(2-(2-(4-(4-(2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamido)phenyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanoate(4)

A mixture ofN-(4-ethynylphenyl)-2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamide(290 mg, 1.0 mmol), 2,5-dioxopyrrolidin-1-yl3-(2-(2-azidoethoxy)ethoxy)propanoate (300 mg, 1.0 mmol), copper (I)iodide (50 mg) and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine(5.0 mg) in chloroform (15 mL) was stirred at room temperatureovernight. The solvent was evaporated and purified by flashchromatography to give a yellow powder (480 mg, 82%).

¹H NMR: (500 MHz, CDCl₃) δ(ppm)): 10.82 (s, 1H), 7.95 (s, 1H), 7.83 (s,4H), 7.56 (d, J=3.3 Hz, 1H), 7.06 (d, J=3.4 Hz, 1H), 6.52 (s, 1H), 4.59(t, J=5.0 Hz, 2H), 3.91 (t, J=5.1 Hz, 2H), 3.83 (t, J=6.0 Hz, 2H), 3.64(s, 4H), 2.86 (t, J=6.0 Hz, 2H), 2.78 (s, 4H), 2.64 (s, 3H), 2.57 (s,3H). ¹³C NMR: (125 MHz, CDCl₃) δ(ppm)): 169.1, 166.9, 162.4, 155.7,147.7, 142.6, 139.6, 139.5, 126.4, 125.6, 120.6, 119.6, 117.2, 108.4,107.0, 105.8, 70.9, 70.6, 69.8, 65.9, 50.5, 32.4, 25.7, 24.8, 18.3.ESI-MS m/z: 590 (M+H⁺), 612 (M+Na⁺).

Example 5 Glucocerebrosidase Activity Assay with Blue Substrate and RedSubstrate

The compounds in DMSO solution 0.5 μL/well were transferred to a black96-well plate (the final titration was 24 nM to 50 μM, 12concentrations, 2 times dilution). 33.5 μL enzyme solution (7.5 nM finalconcentration) was transferred to the wells. After 5 min of incubationat room temperature, the enzyme reaction was initiated by the additionof 33 μL/well blue substrate or 66.5 μL/well red substrate. Finalconcentrations of the blue substrate (4MU-Glc) and red substrate(Res-Glc) were 1.5 mM and 30 μM, respectively. The red substratereaction was measured in the Biotek Synergy H1 multi-mode plate readerwith Ex=573 nm and Em=610 nm at 37° C. every 20 sec for 30 min. The bluesubstrate reaction was terminated by the addition of 33 μL/well stopsolution (1 M NaOH and 1 M glycine mixture, pH 10) after 30 min ofincubation at 37° C. The fluorescence was then measured in the platereader at Ex=365 nm and Em=440 nm.

Example 6 Glucocerebrosidase Activity Assay with Natural Substrate

The compounds in DMSO solution (0.5 μL/well) were transferred to a black96-well plate. The final titration was 24 nM to 50 μM, 12concentrations, 2 times dilution. 33.5 μL enzyme solution wastransferred to the wells (7.5 nM final concentration). After 5 min ofincubation at room temperature, the enzyme reaction was initiated by theaddition of 16 μL/well natural substrate. Final concentration of thenatural substrate (glucosylceramide) was 100 μM. The plate was incubatedfor 30 min at 37° C. and the Amplex Red Glucose/Glucose Oxidase Assaybuffer (50 μL/well) was added. The plate was measured in the BiotekSynergy H1 multi-mode plate reader with Ex=573 nm and Em=610 nm at 37°C. every 20 sec for 30 min.

Example 7 Fluorescence Polarization Assay

The fluorescent probe 3 (25 nL/well, 50 nM final concentration) wastransferred to a 384-well black plate using a Labcyte Echo 550 LiquidHandler system. The 25 μL/well enzyme dilutions with GCase enzymeactivity buffer were added to the plate, which was shaken at roomtemperature in dark for 20 min. The final titration was 5 nM to 10 μM,10 concentrations, 2 times dilution. The fluorescence polarization wasmeasured in Molecular Devices Analyst GT with Ex=535 nm and Em=580 nm, GFactor=1.05.

Example 8 Compound High Throughput Screening (HTS) by FluorescencePolarization

The enzyme in GCase enzyme activity buffer (25 μL/well) was added to a384-well black plate. The fluorescent probe 3 (25 nL/well, 50 nM finalconcentration) was transferred to a 384-well black plate using a LabcyteEcho 550 Liquid Handler system. Compounds in DMSO stock solution (50 nL)were transferred to the plate. The plate was shaken at room temperaturein dark for 20 min. The final concentration was 19.5 nM to 10 KM, 10concentrations, 2 times dilution. The fluorescence polarization wasmeasured in a Molecular Devices Analyst GT with Ex=535 nm and Em=580 nm,G Factor=1.05.

Example 9 Preparation of the Compound-Activated Glucocerebrosidase

To the recombinant wild type enzyme (22 μM, 95 μL, 1 equiv) in 0.1 Mphosphate buffer (pH 7.2) was added 2,5-dioxopyrrolidin-1-yl3-(2-(2-(4-(4-(2,4-dimethylpyrrolo[1,2-a]pyrimidine-8-carboxamido)phenyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanoate(4) in DMSO (0.89 mM, 5 μL, 2 equiv) in one portion, and was vortexedfor 5 sec immediately. At indicated time points, the reaction solution(2 μL) was sampled and diluted (1:3125 dilution) into the assay buffer(50 mM citric acid, 176 mM K₂HPO₄, and 0.01% Tween-20 at pH 5.9). After2 h the reaction solution was dialyzed three times with 0.1 M phosphatebuffer (pH 7.2). The enzyme was adjusted to the same concentration andsampled for activity. The dilution solutions were assayed with threesubstrates, resorufin substrate, 4-MU substrate, and natural substrateusing the method in Example 5 and Example 6 without adding compounds.

Example 10 Synthesis and Testing of Additional Compounds

Additional compounds were prepared and tested according to theprocedures provided in the examples above. Dose-response curves wereprepared to determine IC₅₀ or AC₅₀. Results are shown in Table 1.

TABLE 1 No. Structures MW Ac₅₀ (μM) ¹H NMR ¹³C NMR Mass 1

289 0.14 ¹H NMR: (500 MHz, CDCl₃) δ (ppm): 10.87 (s, 1H), 7.74 (d, J =8.6 Hz, 2H), 7.56 (d, J = 3.3 Hz, 1H), 7.48 (d, J = 8.6 Hz, 2H), 7.07(d, J = 3.3 Hz, 1H), 6.53 (s, 1H), 3.04 (s, 1H), 2.63 (s, 3H), 2.58 (s,3H). ¹³C NMR: (125 MHz, CDCl₃) δ (ppm): 162.5, 155.7, 142.7, 140.3,139.6, 133.0, 119.0, 117.3, 116.0, 108.5, 107.2, 105.6, 84.1, 76.2,24.8,18.3. ESI-MS m/z: 312 (M + Na)⁺. 2

589 3.98 ¹H NMR: (500 MHz, CDCl₃) δ (ppm): 10.82 (s, 1H), 7.95 (s, 1H),7.83 (s, 4H), 7.56 (d, J = 3.3 Hz, 1H), 7.06 (d, J = 3.4 Hz, 1H), 6.52(s, 1H), 4.59 (t, J = 5.0 Hz, 2H), 3.91 (t, J = 5.1 Hz, 2H), 3.83 (t, J= 6.0 Hz, 2H), 3.64 (s, 4H), 2.86 (t, J = 6.0 Hz, 2H), 2.78 (s, 4H),2.64 (s, 3H), 2.57 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃) δ (ppm): 169.1,166.9, 162.4, 155.7, 147.7, 142.6, 139.6, 139.5, 126.4, 125.6, 120.6,119.6, 117.2, 108.4, 107.0, 105.8, 70.9, 70.6, 69.8, 65.9, 50.5, 32.4,25.7, 24.8, 18.3. ESI-MS m/z: 590 (M + H)⁺ 3

463 6.31 ¹H NMR: (500 MHz, CDCl₃) δ (ppm): 10.82 (s, 1H), 7.92 (s, 1H),7.83 (d, J = 8.6 Hz, 2H), 7.80 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 3.2 Hz,1H), 7.05 (d, J = 3.2 Hz, 1H), 6.51 (s, 1H), 4.57 (t, J = 5.0 Hz, 2H),3.90 (t, J = 5.1 Hz, 2H), 3.63-3.54 (m, 4H), 3.45 (t, J = 5.2 Hz, 2H),2.82 (t, J = 5.2 Hz, 2H), 2.63 (s, 3H), 2.56 (s, 3H), 2.15 (br, 2H). ¹³CNMR: (125 MHz, CDCl₃) δ (ppm): 162.5, 155.6, 147.8, 142.6, 139.6, 139.5,128.2, 126.4, 125.5, 120.4, 119.6, 119.3, 117.3, 108.4, 107.1, 105.9,73.5, 70.8, 70.3, 69.7, 50.4, 41.8, 24.8, 18.3. ESI-MS m/z: 464 (M + H)⁺4

875 Kd = 0.79 μM ¹H NMR: (500 MHz, CDCl₃) δ (ppm): 10.84 (s, 1H), 8.35(s, 1H), 8.19 (dd, J = 8.0, 1.2 Hz, 1H), 7.92 (s, 1H), 7.84 (d, J = 8.6Hz, 2H), 7.79 (d, J = 8.6 Hz, 2H), 7.57 (d, J = 3.3 Hz, 1H), 7.24 (d, J= 8.0 Hz, 1H), 7.10 (t, J = 4.9 Hz, 1H), 7.07 (d, J = 3.3 Hz, 1H), 6.55(d, J = 8.0 Hz, 2H), 6.53 (s, 1H), 6.46 (d, J = 2.4 Hz, 2H), 6.35 (dd, J= 8.9, 2.5 Hz, 2H), 4.64 (t, J = 5.1 Hz, 2H), 4.02 (t, J = 5.1 Hz, 2H),3.71-3.61 (m, 8H), 2.96 (s, 12H), 2.64 (s, 3H), 2.59 (s, 3H). ¹³C NMR:(125 MHz, CDCl₃) δ (ppm): 169.2, 166.2, 162.5, 155.7, 153.1, 152.3,147.8, 142.6, 139.7, 139.5, 136.3, 134.4, 128.8, 128.2, 126.4, 125.3,124.8, 123.0, 120.5, 119.7, 117.3, 108.9, 108.5, 107.1, 106.3, 105.8,98.5, 70.6, 70.5, 69.6, 50.4, 40.3, 40.1, 24.8, 18.3. ESI-MS m/z: 898(M + Na)⁺, 876 (M + H)⁺. 5

333 0.8 ¹H NMR (500 MHz, CDCl₃) δ 10.99 (s, 1H), 7.87 (d, J = 8.5 Hz,2H), 7.59 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 3.3 Hz, 1H), 7.08 (d, J =3.3 Hz, 1H), 6.55 (s, 1H), 2.64 (s, 3H), 2.59 (s, 3H). ¹³C NMR: (125MHz, CDCl₃) 162.6, 155.9, 142.8, 142.8, 1397.7, 126.2 (q, J_(C-F) = 3.6Hz), 124.6 (q, J_(C-F) = 33.7 Hz), 124.5 (q, J_(C-F) = 270 Hz), 118.9,117.3, 108.6, 107.3, 105.4, 77.4, 77.1, 76.9, 24.8, 18.3, ESI-MS m/z:356 (M + Na)⁺, 334 (M + H)⁺ 6

302 NA ¹H NMR (500 MHz, CDCl₃) δ 8.80 (s, 1H), 7.51 (d, J = 3.2 Hz, 1H),7.02 (d, J = 3.3 Hz, 1H), 6.47 (s, 1H), 3.77 (s, 4H), 3.67 (d, J = 5.7Hz, 2H), 2.65 (s, 2H), 2.57 (d, J = 10.9 Hz, 10H). ¹³C NMR: (125 MHz,CDCl₃) δ 164.6, 155.1, 142.2, 139.5, 117.3, 108.1, 106.5, 105.9, 67.3,57.9, 53.7, 35.9, 24.9, 18.3. ESI-MS m/z: 303 (M + H)⁺ 7

364 112.2 ¹H NMR (500 MHz, CDCl₃) δ 10.75 (s, 1H), 7.73 (d, J = 8.4 Hz,2H), 7.55 (d, J = 3.2 Hz, 1H), 7.30 (d, J = 8.3 Hz, 2H), 7.05 (d, J =3.2 Hz, 1H), 6.51 (s, 1H), 3.71 (t, J = 4.6 Hz, 4H), 3.48 (s, 2H), 2.61(s, 3H), 2.57 (s, 3H), 2.46 (s, 4H). ¹³C NMR: (125 MHz, CDCl₃) δ 162.3,155.4, 142.5, 139.3, 138.6, 132.0, 129.8, 119.3, 117.1, 108.3, 106.9,105.7, 67.0, 63.1, 53.5, 24.7, 18.2. ESI-MS m/z: 387 (M + Na)⁺, 365 (M +H)⁺ 8

377 39.81 ¹H NMR (500 MHz, CDCl₃) δ 10.74 (s, 1H), 7.72 (d, J = 8.4 Hz,2H), 7.58 (d, J = 3.2 Hz, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.07 (d, J =3.3 Hz, 1H), 6.52 (s, 1H), 3.49 (s, 2H), 2.62 (s, 3H), 2.59 (s, 3H),2.45 (brs, 5H), 2.28 (s, 3H), 1.73 (m, 3H). ¹³C NMR: (125 MHz, CDCl₃)162.3, 155.4, 142.5, 139.2, 138.5, 132.5, 129.8, 119.2, 117.0, 108.2,106.8, 105.6, 62.7, 55.1, 53.0, 46.0, 24.6, 18.1, ESI-MS m/z: 400 (M +Na)⁺, 378 (M + H)⁺ 9

362 70.79 ¹H NMR (500 MHz, CDCl₃) δ 10.71 (s, 1H), 7.71 (d, J = 8.3 Hz,2H), 7.53 (dd, J = 3.1, 0.8 Hz, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.02 (d,J = 2.4 Hz, 1H), 6.48 (s, 1H), 3.46 (s, 2H), 2.59 (s, 3H), 2.54 (s, 3H),2.38 (s, 4H), 1.57 (p, J = 5.6 Hz, 4H), 1.42 (s, 2H). ¹³C NMR: (125 MHz,CDCl₃) 162.3, 155.3, 142.4, 139.2, 138.3, 132.8, 129.9, 119.1, 117.1,108.2, 106.8, 105.7, 63.5, 54.3, 25.9, 24.6, 24.4, 18.1 ESI-MS m/z: 363(M + H)⁺ 10

363 7.08 ¹H NMR (500 MHz, CDCl₃) δ 10.59 (s, 1H), 7.68 (d, J = 8.8 Hz,2H), 7.57 (d, J = 3.2 Hz, 1H), 7.06 (d, J = 3.2 Hz, 1H), 6.96 (d, J =8.9 Hz, 2H), 6.50 (s, 1H), 3.22-3.14 (m, 4H), 2.64-2.55 (m, 10H), 2.36(s, 3H). ¹³C NMR : (125 MHz, CDCl₃) 162.2, 155.3, 147.4, 142.4, 139.3,132.6, 120.7, 117.3, 117.0, 108.2, 106.8, 106.1, 55.3, 50.0, 46.3, 24.8,18.3 ESI-MS (m/z: 364 (M + H)⁺ 11

377 31.62 ¹H NMR (500 MHz, CDCl₃) δ 10.57 (s, 1H), 7.67 (d, J = 7.6 Hz,2H), 7.54 (s, 1H), 7.03 (s, 1H), 6.96 (d, J = 7.6 Hz, 2H), 6.48 (s, 1H),3.19 (s, 4H), 2.76-2.39 (m, 12H), 1.13 (s, 3H). ¹³C NMR: (125 MHz,CDCl₃) 162.2, 155.3, 147.5, 142.4, 139.2, 132.5, 120.7, 117.2, 117.0,108.2, 106.8, 106.0, 53.0, 52.5, 50.0, 24.7, 18.2, 12.1, ESI-MS m/z: 378(M + H)⁺ 12

439 5.01 ¹H NMR (500 MHz, CDCl₃) δ 10.58 (s, 1H), 7.67 (d, J = 8.9 Hz,2H), 7.55 (d, J = 2.9 Hz, 1H), 7.38-7.31 (m, 4H), 7.28 (d, J = 7.0 Hz,1H), 7.04 (t, J = 3.1 Hz, 1H), 6.95 (d, J = 9.0 Hz, 2H), 6.49 (s, 1H),3.58 (s, 2H), 3.24- 3.10 (m, 4H), 2.66- ¹³C NMR: (125 MHz, CDCl₃) 162.2,155.2, 147.5, 142.4, 139.2, 138.1, 132.5, 129.3, 128.3, 127.2, 120.7,117.2, 117.0, 108.2, 106.8, 106.0, ESI-MS m/z: 440 (M + H)⁺ 2.61 (m,4H), 2.59 (s, 63.2, 53.2, 50.0, 3H), 2.56 (s, 3H). 24.7, 18.3 13

355 0.79 ¹H NMR (500 MHz, CDCl₃) δ 10.71 (s, 1H), 7.70 (d, J = 8.3 Hz,2H), 7.56 (d, J = 3.2 Hz, 1H), 7.31-7.26 (m, 2H), 7.24- 7.13 (m, 5H),7.04 (d, J = 3.2 Hz, 1H), 6.50 (s, 1H), 3.98 (s, 2H), 2.59 (s, 3H), 2.56(s, 3H). ¹³C NMR: (125 MHz, CDCl₃) 162.4, 155.4, 142.5, 141.6, 139.4,137.7, 135.7, 129.5, 129.0, 128.5, 126, 119.7, 117.2, 108.3, 106.9,105.9, 41.5, 24.7, 18.2 ESI-MS m/z: 378 (M + Na)⁺, 356 (M + H)⁺ 14

378 31.62 ¹H NMR (500 MHz, CDCl₃) δ 10.71 (s, 1H), 7.70 (d, J = 8.2 Hz,2H), 7.59-7.53 (m, 1H), 7.19 (d, J = 8.2 Hz, 2H), 7.08- 7.02 (m, 1H),6.51 (s, 1H), 3.75 (t, J = 4.4 Hz, 4H), 2.83-2.76 (m, 2H), 2.61 (s, 3H),2.60 (s, 2H), 2.57 (s, 3H), 2.53 (s, 4H). ¹³C NMR: (125 MHz, CDCl₃)162.4, 155.4, 142.5, 139.4, 137.8, 134.7, 129.1, 119.7, 117.3, 108.3,106.9, 105.9, 67.1, 61.1, 53.8, 32.9, 24.8, 18.3 ESI-MS m/z: 379 (M +H)⁺ 15

376 39.81 ¹H NMR (500 MHz, CDCl₃) δ 10.70 (s, 1H), 7.69 (d, J = 8.2 Hz,2H), 7.57 (d, J = 3.2 Hz, 1H), 7.19 (d, J = 8.2 Hz, 2H), 7.06 (d, J =3.2 Hz, 1H), 6.51 (s, 1H), 2.83-2.76 (m, 2H), 2.61 (s, 3H), 2.58 (s,3H), 2.56-2.39 (m, 6H), 1.68-1.57 (m, 4H), 1.46 (brs, 2H). ¹³C NMR: (125MHz, CDCl₃) 162.4, 155.4, 142.5, 139.4, 137.6, 135.3, 129.2, 119.6,117.3, 108.3, 106.9, 106.0, 61.7, 54.7, 33.2, 26.1, 24.8, 24.6, 18.3ESI-MS m/z: 377 (M + H)⁺ 16

391 44.67 ¹H NMR (500 MHz, CDCl₃) δ 10.72 (s, 1H), 7.64 (d, J = 8.1 Hz,2H), 7.48 (d, J = 2.9 Hz, 1H), 7.16 (d, J = 8.1 Hz, 2H), 7.05 (d, J =3.1 Hz, 1H), 6.51 (s, 1H), 2.98-2.64 (m, 12H), 2.59 (s, 3H), 2.55 (s,6H). ¹³C NMR: (125 MHz, CDCl₃) 162.5, 155.8, 142.7, 139.5, 137.8, 133.9,129.1, 119.9, 117.0, 108.5, 107.1, 105.4, 59.4, 53.9, 51.1, 44.6, 32.3,24.7, 18.2. ESI-MS m/z: 392 (M + H)⁺ 17

308 3.55 ¹H NMR (500 MHz, CD₃OD) δ 7.55 (d, J = 8.9 Hz, 2H), 7.44 (d, J= 3.2 Hz, 1H), 7.34 (d, J = 3.2 Hz, 1H), 6.85 (d, J = 8.9 Hz, 2H), 6.77(s, 1H), 2.94 (s, 6H), 2.66 (s, 3H), 2.65 (s, 3H). ¹³C NMR (500 MHz,CD₃OD) 164.6, 158.0, 149.2, 145.2, 141.0, 130.5, 122.3, 117.0, 114.9,109.5, 109.0, 105.3, 41.5, 24.6, 18.0, ESI-MS m/z: 309 (M + H)⁺ 18

348 1.58 ¹H NMR (500 MHz, CDCl₃) δ 10.58 (s, 1H), 7.66 (d, J = 8.9 Hz,2H), 7.58 (d, J = 3.2 Hz, 1H), 7.07 (d, J = 3.2 Hz, 1H), 6.97 (d, J =8.9 Hz, 2H), 6.51 (s, 1H), 3.11 (t, J = 5.5 Hz, 4H), 2.61 (s, 3H), 2.58(s, 3H), 1.77-1.69 (m, 4H), 1.64-1.53 (m, 2H). ¹³C NMR: (125 MHz, CDCl₃)162.2, 155.2, 148.6, 142.4, 139.3, 132.2, 120.7, 117.6, 117.3, 108.2,106.8, 106.1, 51.7, 261.1, 24.8, 24.4, 18.3, ESI-MS m/z: 349 (M + H)⁺ 19

350 1.41 ¹H NMR (500 MHz, CDCl₃) δ 10.62 (s, 1H), 7.70 (d, J = 8.9 Hz,2H), 7.58 (d, J = 3.2 Hz, 1H), 7.07 (d, J = 3.2 Hz, 1H), 6.94 (d, J =8.9 Hz, 2H), 6.52 (s, 1H), 3.88 (t, J = 4.7 Hz, 4H), 3.13 (t, J = 4.7Hz, 4H), 2.62 (s, 3H), 2.59 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃) 162.1,155.2, 147.2, 142.4, 139.2, 132.8, 120.7, 117.2, 116.6, 108.2, 106.7,105.9, 67.0, 50.2, 24.7, 18.2, ESI-MS m/z: 373 (M + Na)⁺, 351 (M + H)⁺20

322 14.13 ¹H NMR (500 MHz, CDCl₃) δ 10.84 (s, 1H), 7.75 (d, J = 8.3 Hz,2H), 7.54 (d, J = 3.2 Hz, 1H), 7.35 (d, J = 8.3 Hz, 2H), 7.09 (d, J =3.3 Hz, 1H), 6.55 (s, 1H), 3.64 (s, 2H), 2.63 (s, 3H), 2.60 (s, 3H),2.42 (s, 6H). ¹³C NMR: (125 MHz, CDCl₃) 162.7, 155.8, 142.7, 139.6,139.3, 130.5, 119.7, 117.2, 108.5, 107.2, 105.5, 63.3, 44.5, 24.8, 18.3,ESI-MS m/z: 323 (M + H)⁺ 21

316 39.8 ¹H NMR (500 MHz, CDCl₃) δ 11.57 (s, 1H), 8.46 (d, J = 8.5 Hz,1H), 8.43 (d, J = 5.7 Hz, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.68 (t, J =7.1 Hz, 1H), 7.63 (d, J = 3.3 Hz, 1H), 7.59 (t, J = 7.2 Hz, 1H), 7.44(d, J = 5.7 Hz, 1H), 7.08 (d, J = 3.3 Hz, 1H), 6.54 (s, 1H), 2.65 (s,3H), 2.58 (s, 3H).. ¹³C NMR: (125 MHz, CDCl₃) 161.9, 155.9, 150.5,142.9, 141.5, 140.0, 137.8, 130.2, 127.1, 126.8, 124.6, 122.1, 117.7,117.5, 108.6, 107.3, 106.0, 24.5, 18.3, ESI-MS m/z: 317 (M + H)⁺ 22

266 25.12 ¹H NMR (500 MHz, CDCl₃) δ 10.97 (s, 1H), 8.47 (dd, J = 5.0,1.3 Hz, 2H), 7.64 (dd, J = 4.9, 1.4 Hz, 2H), 7.51 (d, J = 3.3 Hz, 1H),7.05 (d, J = 3.3 Hz, 1H), 6.53 (s, 1H), 2.62 (s, 3H), 2.57 (s, 3H). ¹³CNMR: (125 MHz, CDCl₃) 218.4, 162.9, 156.2, 150.5, 146.4, 143.0, 139.9,117.2, 113.4, 108.7, 107.5, 105.1, 24.8, 18.3, ESI-MS m/z: 267 (M + H)⁺23

266 22.39 ¹H NMR (500 MHz, CDCl₃) δ 11.16 (s, 1H), 8.44 (d, J = 8.4 Hz,1H), 8.34 (d, J = 4.7 Hz, 1H), 7.70 (s, 1H), 7.55 (d, J = 3.2 Hz, 1H),7.06 (d, J = 3.2 Hz, 1H), 7.02-6.92 (m, 1H), 6.51 (s, 1H), 2.66 (s, 3H),2.56 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃) 162.7, 156.3, 152.9, 148.0,142.5, 139.9, 138.0, 118.7, 117.2, 114.4, 108.7, 107.2, 105.2, 24.9,18.2, ESI-MS m/z: 289 (M + Na)⁺, 267 (M + H)⁺ 24

341 1.41 ¹H NMR (500 MHz, CDCl₃) δ 10.84 (s, 1H), 7.87 (d, J = 8.6 Hz,2H), 7.67-7.54 (m, 5H), 7.47- 7.39 (m, 2H), 7.32 (t, J = 7.4 Hz, 1H),7.09 (d, J = 3.2 Hz, 1H), 6.54 (s, 1H), 2.65 (s, 3H), 2.60 (s, 3H) ¹³CNMR: (125 MHz, CDCl₃) 162.5, 155.6, 142.6, 141.0, 139.4, 139.1, 135.6,128.8, 127.6, 126.8, 119.7, 117.2, 117.2, 108.4, 107.0, 105.8, 24.8,18.2, ESI-MS m/z: 364 (M + Na)⁺, 342 (M + H)⁺ 25

334 0.51 ¹H NMR (500 MHz, CDCl₃) δ 10.47 (s, 1H), 7.63 (d, J = 8.9 Hz,2H), 7.57 (d, J = 3.2 Hz, 1H), 7.05 (d, J = 3.3 Hz, 1H), 6.59 (d, J =8.9 Hz, 2H), 6.49 (s, 1H), 3.29 (t, J = 6.5 Hz, 4H), 2.60 (s, 3H), 2.57(s, 3H), 2.03-1.98 (m, 4H). ¹³C NMR: (125 MHz, CDCl₃) 162.0, 155.1,144.8, 142.3, 139.1, 128.7, 121.5, 117.3, 111.9, 108.1, 106.6, 106.3,77.4, 77.1, 76.9, 48.0, 25.5, 24.7, 18.3, ESI-MS m/z: 335 (M + H)⁺ 26

305 1.26 ¹H NMR (500 MHz, d6- DMSO) δ 12.17 (s, 1H), 11.52 (s, 1H),7.57-7.50 (m, 2H), 7.49-7.39 (m, 2H), 7.10 (m, 2H), 6.96 (s, 1H), 2.67(s, 3H), 2.65 (s, 3H). ¹³C NMR: (125 MHz, d6-DMSO) 161.5, 157.9, 147.1,145.2, 141.1, 140.3, 133.3, 121.6, 120.9, 117.1, 116.1, 111.9, 110.1,109.5, 102.6, 24.8, 18.1, ESI-MS m/z: 306 (M + H)⁺ 27

271 15.85 ¹H NMR (500 MHz, CDCl₃) δ 8.65 (d, J = 7.0 Hz, 1H), 7.52 (d, J= 3.2 Hz, 1H), 7.01 (d, J = 3.2 Hz, 1H), 6.45 (s, 1H), 4.14-4.01 (m,1H), 2.55 (s, 6H), 2.10-1.97 (m, 2H), 1.81-1.70 (m, 2H), 1.66-1.53 (m,1H), 1.53- 1.26 (m, 5H). ¹³C NMR: (125 MHz, CDCl₃) 163.6, 154.9, 142.1,139.3, 117.2, 107.9, 106.3, 106.0, 47.1, 33.3, 26.0, 24.8, 24.6, 18.2,ESI-MS m/z: 294 (M + Na)⁺, 272 (M + H)⁺ 28

305 3.98 ¹H NMR (50 0MHz, CDCl₃) δ 8.94 (d, J = 7.8 Hz, 1H), 7.55 (d, J= 3.3 Hz, 1H), 7.45 (d, J = 6.9 Hz, 1H), 7.30-7.19 (m, 3H), 7.04 (d, J =3.3 Hz, 1H), 6.44 (s, 1H), 5.74 (q, J = 8.0 Hz, 1H), 3.04 (ddd, J =15.7, 8.7, 2.5 Hz, 1H), 2.98-2.89 (m, 1H), 2.80-2.70 (m, 1H), 25.6 (s,3H), 2.44 (s, 3H), 2.04-1.92 (m, 1H). ¹³C NMR: (125 MHz, CDCl₃) 164.6,155.2, 144.8, 143.3, 142.1, 139.6, 127.4, 126.6, 124.7, 124.2, 117.2,108.1, 106.5, 105.5, 54.5, 34.9, 30.5, 24.7, 18.2, ESI-MS m/z: 329 (M +Na)⁺, 306 (M + H)⁺ 29

305 2.51 ¹H NMR (500 MHz, CDCl₃) δ 8.94 (d, J = 7.2 Hz, 1H), 7.52 (d, J= 3.3 Hz, 1H), 7.27 (m, 2H), 7.20-7.16 (m, 2H), 7.01 (d, J = 3.3 Hz,1H), 6.42 (s, 1H), 5.08-4.93 (m, 1H), 3.45 (dd, J = 15.8, 7.3 Hz, 2H),3.00 (dd, J = 15.8, 6.0 Hz, 2H), 2.54 (s, 3H), 2.42 (s, 3H). ¹³C NMR:(125 MHz, CDCl₃) 164.3, 155.1, 142.1, 141.7, 139.4, 126.6, 124.8, 117.1,108.0, 106.4, 105.7, 50.4, 40.6, 24.6, 18.2, ESI-MS m/z: 328 (M + Na)⁺,306 (M + H)⁺ 30

309 2.82 ¹H NMR (500 MHz, CDCl₃) δ 10.73 (s, 1H), 7.72 (d, J = 8.3 Hz,2H), 7.55 (d, J = 3.2 Hz, 1H), 7.21 (d, J = 8.3 Hz, 2H), 7.05 (d, J =3.2 Hz, 1H), 6.51 (s, 1H), 3.85 (t, J = 6.6 Hz, 2H), 2.86 (t, J = 6.6Hz, 2H), 2.61 (s, 3H), 2.57 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃) 162.4,155.5, 142.6, 139.4, 138.1, 132.9, 129.5, 119.8, 117.2, 108.4, 107.0,105.8, 63.9, 38.8, 24.8, 18.3, ESI-MS m/z: 332 (M + Na)⁺, 310 (M + H)⁺31

322 0.40 ¹H NMR (500 MHz, d6- DMSO) δ 12.14 (s, 1H), 8.00 (d, J = 7.5Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.58 (s, 1H), 7.49-7.40 (m, 2H), 7.31(t, J = 6.9 Hz, 1H), 7.01 (s, 1H), 2.68 (s, 3H), 2.67 (s, 3H). ¹³C NMR:(125 MHz, d6-DMSO) 161.1, 158.3, 158.0, 149.1, 145.4, 140.7, 132.1,126.5, 123.7, 122.1, 120.7, 116.1, 110.6, 109.8, 101.8, 24.8, 18.1,ESI-MS m/z: 345 (M + Na)⁺, 323 (M + H)⁺ 32

362 50 ¹H NMR (500 MHz, CDCl₃) δ 8.67 (d, J = 6.1 Hz, 1H), 7.50 (d, J =3.2 Hz, 1H), 7.37-7.30 (m, 4H), 7.29-7.24 (m, 1H), 7.01 (d, J = 3.2 Hz,1H), 6.46 (s, 1H), 4.20-4.09 (m, 1H), 3.56 (s, 2H), 2.90-2.82 (m, 2H),2.55 (s, 3H), 2.54 (s, 3H), 2.25-2.35 (m, 2H), 2.11- 2.03 (m, 2H),1.75-1.66 (m, 2H). ¹³C NMR: (125 MHz, CDCl₃) 163.8, 155.1, 142.1, 139.4,129.4, 128.3, 127.2, 117.2, 108.0, 106.4, 105.8, 63.4, 52.3, 45.3, 32.4,24.7, 18.2, ESI-MS m/z: 363 (M + H)⁺ 33

286 NA ¹H NMR (500 MHz, d6- DMSO) δ 7.36 (d, J = 3.2 Hz, 1H), 7.22 (d, J= 3.2 Hz, 1H), 6.79 (s, 1H), 3.99 (m, 1H), 3.43 (m, 2H), 3.10 (m, 2H),2.76 (s, 3H), 2.58 (s, 3H), 2.50 (s, 3H), 2.24-2.06 (m, 2H), 1.76-1.59(m, 2H). ¹³C NMR: (125 MHz, d6-DMSO) 162.8, 155.8, 143.7, 138.7, 115.6,108.1, 104.1, 52.8, 43.1, 42.5, 29.6, 24.3, 17.6, ESI-MS m/z: 287 (M +H)⁺ 34

314 30 ¹H NMR (500 MHz, CD₃OD) δ 7.40 (d, J = 3.3 Hz, 1H), 7.34 (d, J =3.3 Hz, 1H), 6.79 (s, 1H), 4.22 (brs, 1H), 3.59 (m, 2H), 3.28 (m, 1H),2.67 (s, 3H), 2.60 (s, 3H), 2.43 (brs, 2H), 1.90 (brs, 2H), 1.42 (d, J =6.7 Hz, 6H). ¹³C NMR: (125 MHz, CD₃OD) 166.6, 158.1, 145.3, 141.4,116.9, 109.5, 109.0, 104.5, 59.5, 45.5, 31.1, 24.6, 17.9, 17.0, ESI-MSm/z: 315 (M + H)⁺ 35

348 22.39 ¹H NMR (500 MHz, CDCl₃) δ 8.75 (d, J = 7.8 Hz, 1H), 7.51 (d, J= 3.2 Hz, 1H), 7.30-7.25 (m, 2H), 7.02 (d, J = 3.2 Hz, 1H), 6.99 (d, J =8.0 Hz, 2H), 6.85 (t, J = 7.3 Hz, 1H), 6.46 (s, 1H), 4.36- 4.19 (m, 1H),3.67-3.52 (m, 2H), 3.13-3.00 (m, 2H), 2.55 (s, 3H), 2.52 (s, 3H),2.29-2.14 (m, 2H), 1.87-1.74 (m, 2H). ¹³C NMR: (125 MHz, CDCl₃) 163.8,155.2, 151.7, 142.2, 139.4, 129.2, 119.6, 117.1, 116.7, 108.0, 106.4,105.7, 48.5, 45.3, 32.2, 24.8, 18.2, ESI-MS m/z: 371 (M + Na)⁺, 349 (M +H)⁺ 36

331 107 ¹H NMR (500 MHz, CDCl₃) δ 10.90 (s, 1H), 7.89 (d, J = 8.7 Hz,2H), 7.82 (s, 1H), 7.56 (d, J = 3.2 Hz, 1H), 7.36 (d, J = 8.7 Hz, 2H),7.19 (s, 1H), 7.08 (d, J = 3.2 Hz, 1H), 6.54 (s, 1H), 2.64 (s, 3H), 2.59(s, 3H). ¹³C NMR: (125 MHz, CDCl₃) 162.5, 155.8, 142.8, 139.6, 139.2,135.7, 132.2, 130.2, 122.3, 120.4, 118.6, 117.2, 108.5, 107.2, 105.5,24.8, 18.3, ESI-MS m/z: 332 (M + H)⁺ 37

314 40 ¹H NMR (500 MHz, CD₃OD) δ 7.39 (d, J = 3.3 Hz, 1H), 7.33 (d, J =3.3 Hz, 1H), 6.78 (s, 1H), 4.31 (t, J = 2.7 Hz, 1H), 3.42-3.35 (m, 1H),2.92 (s, 6H), 2.67 (s, 3H), 2.63 (s, 3H), 2.19 (d, J = 10.4 Hz, 2H),2.10 (d, J = 9.1 Hz, 2H), 1.92-1.79 (m, 4H). ¹³C NMR: (125 MHz, CD₃OD)166.4, 158.1, 145.3, 141.3, 116.8, 109.4, 108.9, 105.0, 65.8, 44.3,40.3, 29.6, 24.9, 23.0, 17.9, ESI-MS m/z: 315 (M + H)⁺ 38

365 0.79 (TC) ¹H NMR (500 MHz, CDCl₃) δ 10.87 (s, 1H), 7.77 (d, J = 8.6Hz, 2H), 7.58-7.49 (m, 5H), 7.39- 7.28 (m, 3H), 7.06 (d, J = 3.3 Hz,1H), 6.52 (s, 1H), 2.63 (s, 3H), 2.57 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃)162.5, 155.8, 142.8, 140.0, 139.6, 132.6, 131.7, 128.5, 128.1, 123.8,119.1, 117.3, 108.6, 107.2, 105.7, 90.0, 88.6, 24.9, 18.4, ESI-MS m/z:388 (M + Na)⁺, 366 (M + H)⁺ 39

341 39.81 ¹H NMR (400 MHz, d6- DMSO) δ 11.80 (s, 1H), 7.51 (d, J = 3.4Hz, 1H), 7.37 (d, J = 3.4 Hz, 1H), 6.92 (s, 1H), 3.80 (s, 2H), 2.98 (t,J = 5.5 Hz, 2H), 2.77 (t, J = 5.5 Hz, 2H), 2.64 (s, 3H), 2.60 (s, 3H),2.56 (s, 3H). ¹³C NMR: (100 MHz, d6-DMSO) 160.0, 157.4, 144.7, 142.0,139.8, 117.2, 115.5, 109.7, 109.0, 101.5, 51.6, 51.0, 43.9, 25.4, 24.3,17.6 ESI-MS m;z 342 (M + H)⁺ 40

320 ¹H NMR (400 MHz, d6- DMSO) δ 10.86 (s, 1H), 7.91 (s, 1H), 7.62 (dd,J = 8.2, 1.7 Hz, 1H), 7.46 (d, J = 3.3 Hz, 1H), 7.38- 7.34 (m, 2H), 6.89(s, 1H), 4.52 (s, 2H), 4.46 (s, 2H), 2.96 (s, 3H), 2.64 (s, 3H), 2.63(s, 3H). ¹³C NMR: (100 MHz, d6-DMSO) 161.4, 156.5, 144.2, 139.4, 138.9,136.4, 129.4, 123.2, 118.5, 115.7, 112.8, 108.7, 108.5, 103.9, 59.8,59.4, 40.7, 24.2, 17.6, ESI-MS m/z: 321 (M + H)⁺ 41

356 39.91 ¹H NMR (500 MHz, CDCl₃) δ 12.14 (s, 1H), 7.80 (d, J = 1.9 Hz,1H), 7.73 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 3.4 Hz, 1H), 7.24 (d, J =2.0 Hz, 1H), 7.14 (d, J = 3.4 Hz, 1H), 6.63 (d, J = 1.2 Hz, 1H), 2.73(s, 3H), 2.64 (s, 3H). ¹³C NMR: (125 MHz, CDCl3) 161.8, 160.3, 157.6,150.3, 143.1, 140.9, 131.8, 131.0, 123.6, 122.0, 120.5, 117.2, 109.4,108.2, 103.1, 24.9, 18.3, ESI-MS m/z: 379 (M + Na)⁺, 357 (M + H)⁺ 42

340 50.18 ¹H NMR (500 MHz, CDCl₃) δ 12.12 (s, 1H), 7.77-7.70 (m, 1H),7.58 (s, 1H), 7.50 (d, J = 9.8 Hz, 1H), 7.12 (s, 1H), 7.03 (t, J = 8.8Hz, 1H), 6.61 (s, 1H), 2.72 (s, 3H), 2.62 (s, 3H). ¹³C NMR: (125 MHz,CDCl₃) 163.0, 161.7, 161.1, 160.9, 157.5, 150.4, 143.1, 140.9, 128.0,121.9, 121.9, 117.2, 111.6, 111.4, 109.4, 108.2, 107.2, 107.0, 103.2,24.9, 18.3, ESI-MS m/z: 363 (M + Na)⁺, 341 (M + H)⁺ 43

336 0.20 ¹H NMR (500 MHz, CDCl₃) δ 12.06 (s, 1H), 7.67 (d, J = 7.7 Hz,1H), 7.58 (d, J = 3.3 Hz, 1H), 7.23 (d, J = 7.2 Hz, 1H), 7.18 (t, J =7.5 Hz, 1H), 7.11 (d, J = 3.3 Hz, 1H), 6.58 (s, 1H), 2.73 (s, 3H), 2.72(s, 3H), 2.60 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃) 161.8, 157.8, 157.2,143.0, 140.7, 132.4, 130.5, 126.6, 123.2, 118.7, 117.2, 109.2, 108.0,103.4, 24.9, 18.3, 18.1, ESI-MS m/z: 359 (M + Na)⁺, 337 (M + H)⁺ 44

301 22.39 ¹H NMR (500 MHz, CDCl₃) δ 12.09 (s, 1H), 7.56 (d, J = 3.4 Hz,1H), 7.12 (d, J = 3.4 Hz, 1H), 6.61 (s, 1H), 3.08 (q, J = 7.6 Hz, 2H),2.68 (s, 3H), 2.62 (s, 3H), 1.43 (t, J = 7.6 Hz, 3H). ¹³C NMR: (125 MHz,CDCl₃) 166.5, 161.0, 158.9, 157.5, 143.1, 141.0, 117.2, 109.3, 108.1,102.9, 24.7, 23.7, 18.3, 14.3, ESI-MS m/z: 324 (M + Na)⁺, 302 (M + H)⁺45

317 35.48 ¹H NMR (500 MHz, CDCl₃) δ 11.64 (s, 1H), 9.33 (s, 1H), 8.01(d, J = 8.4 Hz, 1H), 7.86-7.80 (m, 2H), 7.68 (d, J = 3.3 Hz, 1H), 7.48(t, J = 7.1 Hz, 1H), 7.11 (d, J = 3.3 Hz, 1H), 6.57 (s, 1H), 2.72 (s,3H), 2.62 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃) 162.1, 161.5, 156.3, 155.5,151.5, 142.7, 140.1, 134.3, 127.7, 127.3, 125.5, 122.2, 117.8, 108.7,107.4, 105.7, 24.9, 18.3, ESI-MS m/z: 340 (M + Na)⁺, 318 (M + H)⁺ 46

334 0.45 ¹H NMR (500 MHz, CDCl₃) δ 11.14 (s, 1H), 8.95 (s, 1H), 7.60 (s,1H), 7.09 (s, 1H), 6.54 (s, 1H), 6.52 (s, 1H), 2.73 (s, 6H), 2.60 (s,6H). ¹³C NMR: (125 MHz, CDCl₃) 161.7, 156.2, 155.6, 144.6, 142.4, 139.8,138.4, 136.4, 117.1, 111.7, 108.3, 108.2, 106.9, 105.8, 24.8, 24.6,18.3, 16.7, ESI-MS m/z: 357 (M + Na)⁺, 335 (M + H)⁺ 47

352 Inhibitor ¹H NMR (500 MHz, CDCl₃) δ 11.96 (s, 1H), 7.71 (d, J = 8.8Hz, 1H), 7.55 (d, J = 3.2 Hz, 1H), 7.32 (d, J = 2.3 Hz, 1H), 7.09 (d, J= 3.2 Hz, 1H), 7.02 (dd, J = 8.8, 2.4 Hz, 1H), 6.56 (s, 1H), 3.88 (s,3H), 2.69 (s, 3H), 2.58 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃) 161.7, 157.3,156.9, 156.4, 143.4, 143.0, 140.7, 133.8, 121.2, 117.1, 114.8, 109.2,108.0, 104.4, 103.3, 55.9, 24.8, 18.2, ESI-MS m/z: 375 (M + Na)⁺, 353(M + H)⁺ 48

336 0.20 ¹H NMR (500 MHz, CDCl₃) δ 12.02 (s, 1H), 7.71 (d, J = 8.2 Hz,1H), 7.64 (s, 1H), 7.60 (d, J = 3.3 Hz, 1H), 7.23 (dd, J = 8.2, 1.1 Hz,1H), 7.13 (d, J = 3.4 Hz, 1H), 6.61 (s, 1H), 2.72 (s, 3H), 2.62 (s, 3H),2.48 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃) 161.7, 158.2, 157.3, 147.1,143.0, 140.8, 133.2, 132.7, 127.4, 121.2, 120.2, 117.2, 109.3, 108.0,103.4, 24.9, 21.5, 18.3, ESI-MS m/z: 359 (M + Na)⁺, 337 (M + H)⁺ 49

298 39.81 ¹H NMR (500 MHz, CDCl₃) δ 11.05 (s, 1H), 7.91 (s, 1H), 7.61(d, J = 3.3 Hz, 1H), 7.07 (d, J = 3.3 Hz, 1H), 6.52 (s, 1H), 4.12 (t, J= 7.1 Hz, 2H), 2.62 (s, 3H), 2.58 (s, 3H), 1.97 (m, 2H), 0.96 (t, J =7.4 Hz, 3H). ¹³C NMR: (125 MHz, CDCl₃) 161.0, 157.4, 156.0, 142.5,141.7, 139.9, 117.7, 108.6, 107.1, 105.2, 51.7, 24.7, 23.1, 18.3, 11.1,ESI-MS m/z: 299 (M + H)⁺ 50

391 10 ¹H NMR (500 MHz, CDCl₃) δ 10.57 (s, 1H), 7.66 (d, J = 8.9 Hz,2H), 7.57 (d, J = 3.2 Hz, 1H), 7.05 (d, J = 3.2 Hz, 1H), 6.96 (d, J =8.9 Hz, 2H), 6.50 (s, 1H), 3.73-3.61 (m, 2H), 2.74-2.64 (m, 2H), 2.60(s, 3H), 2.57 (s, 3H), 2.33 (s, 6H), 2.30- 2.22 (m, 1H), 1.93 (d, J =12.4 Hz, 2H), 1.68 (m, 2H). ¹³C NMR: (125 MHz, CDCl₃) 162.2, 155.3,147.4, 142.4, 139.3, 132.4, 120.7, 117.6, 117.3, 108.2, 106.8, 106.1,62.2, 50.2, 41.8, 28.4, 24.7, 18.3, ESI-MS m/z: 392 (M + H)⁺ 51

285 15.85 ¹H NMR (500 MHz, CDCl₃) δ 9.00 (s, 1H), 7.53 (d, J = 3.3 Hz,1H), 7.20 (dd, J = 5.1, 1.2 Hz, 1H), 7.10-7.04 (m, 1H), 7.00 (d, J = 3.3Hz, 1H), 6.95 (dd, J = 5.1, 3.5 Hz, 1H), 6.43 (s, 1H), 4.90 (d, J = 5.7Hz, 2H), 2.53 (s, 3H), 2.50 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃) 164.3,155.4, 143.0, 142.2, 139.6, 126.8, 125.2, 124.5, 117.2, 108.1, 106.5,105.3, 38.0, 24.6, 18.2, ESI-MS m/z: 308 (M + Na)⁺, 286 (M + H)⁺ 52

306 0.20 ¹H NMR (500 MHz, CDCl₃) δ 10.77 (s, 1H), 7.78 (d, J = 8.8 Hz,2H), 7.56 (d, J = 3.3 Hz, 1H), 7.06 (d, J = 3.3 Hz, 1H), 7.01 (d, J =8.8 Hz, 2H), 6.52 (s, 1H), 2.62 (s, 3H), 2.58 (s, 3H). ¹³C NMR: (125MHz, CDCl₃) 162.4, 155.6, 142.7, 139.5, 136.9, 134.2, 120.8, 119.5,117.3, 108.4, 107.1, 105.7, 24.8, 18.3, ESI-MS m/z: 329 (M + Na)⁺, 307(M + H)⁺ 53

369 0.14 ¹H NMR (500 MHz, CDCl₃) δ 11.07 (s, 1H), 7.89 (d, J = 9.0 Hz,2H), 7.87 (d, J = 9.0 Hz, 2H), 7.80 (d, J = 7.1 Hz, 2H), 7.60-7.53 (m,2H), 7.49 (t, J = 7.6 Hz, 2H), 7.09 (d, J = 3.3 Hz, 1H), 6.56 (s, 1H),2.65 (s, 3H), 2.60 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃) 218.4, 195.8,162.6, 156.0, 143.9, 142.9, 139.8, 138.4, 132.0, 131.9, 131.7, 129.9,128.3, 118.5, 117.3, 108.6, 107.4, 105.6, 24.8, 18.3, ESI-MS m/z: 392(M + Na)⁺, 370 (M + H)⁺ 54

357 0.56 ¹H NMR (500 MHz, CDCl₃) δ 10.73 (s, 1H), 7.76 (d, J = 8.9 Hz,2H), 7.58 (d, J = 3.2 Hz, 1H), 7.35-7.28 (m, 2H), 7.09- 7.03 (m, 4H),7.01 (d, J = 7.9 Hz, 2H), 6.52 (s, 1H), 2.62 (s, 3H), 2.59 (s, 3H). ¹³CNMR: (125 MHz, CDCl₃) 162.4, 158.3, 155.5, 152.2, 142.6, 139.4, 135.6,129.7, 122.7, 121.0, 120.1, 118.1, 117.3, 108.4, 107.0, 105.9, 24.8,18.3, ESI-MS m/z: 380 (M + Na)⁺, 358 (M + H)⁺ 55

355 NA ¹H NMR (500 MHz, CDCl₃) δ 10.82 (s, 1H), 7.85 (d, J = 8.5 Hz,2H), 7.60 (s, 2H), 7.58 (s, 1H), 7.51 (d, J = 8.0 Hz, 2H), 7.24 (d, J =7.9 Hz, 2H), 7.08 (d, J = 3.2 Hz, 1H), 6.53 (s, 1H), 2.64 (s, 3H), 2.59(s, 3H), 2.40 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃) 162.4, 155.5, 142.6,139.5, 138.8, 138.2, 136.6, 135.7, 129.5, 127.4, 126.7, 119.8, 117.4,108.4, 107.0, 106.0, 24.8, 21.2, 18.3, ESI-MS m/z: 378 (M + Na)⁺. 56

353 15.85 ¹H NMR (500 MHz, d6- DMSO) δ 12.40 (s, 1H), 10.60 (s, 1H),7.65 (d, J = 9.0 Hz, 2H), 7.43 (d, J = 3.2 Hz, 1H), 7.34 (d, J = 3.2 Hz,1H), 6.94 (d, J = 9.0 Hz, 2H), 6.84 (s, 1H), 4.17 (t, J = 6.0 Hz, 2H),2.70 (t, J = 6.0 Hz, 2H), 2.63 (s, 3H), 2.61 (s, 3H). ¹³C NMR: (125 MHz,d6-DMSO) 172.3, 161.0, 156.1, 153.9, 143.9, 138.6, 132.7, 120.1, 115.7,114.7, 108.4, 108.3, 104.2, 63.8, 34.2, 24.2, 17.5, ESI-MS m/z: 352 (M −H)⁻ 57

432 7.08 Activator ¹H NMR (500 MHz, d6- DMSO) δ 12.10 (brs, 1H), 10.86(s, 1H), 8.53 (s, 1H), 7.84 (s, 4H), 7.49 (d, J = 3.2 Hz, 1H), 7.38 (d,J = 3.3 Hz, 1H), 6.91 (s, 1H), 4.42 (t, J = 6.9 Hz, 2H), 2.66 (s, 6H),2.29 (s, 2H), 1.96-1.83 (m, 2H), 1.58-1.45 (m, 2H). ¹³C NMR: (125 MHz,d6-DMSO) 161.3, 156.4, 146.2, 144.1, 138.9, 138.9, 125.8, 125.3, 120.6,118.9, 115.8, 108.7, 108.5, 104.1, 49.2, 32.9, 29.1, 24.3, 21.4, 17.6,ESI-MS m/z: 455 (M + Na)⁺, 433 (M + H)⁺ 58

460 1.78 Activator ¹H NMR (500 MHz, d6- DMSO) δ 12.00 (s, 1H), 10.84 (s,1H), 8.52 (s, 1H), 7.83 (s, 4H), 7.46 (d, J = 3.3 Hz, 1H), 7.37 (d, J =3.3 Hz, 1H), 6.87 (s, 1H), 4.39 (t, J = 7.1 Hz, 2H), 2.65 (s, 3H), 2.64(s, 3H), 2.22 (s, 2H), 1.91- 1.84 (m, 2H), 1.55-1.46 (m, 2H), 1.37-1.25(m, 4H). ¹³C NMR: (125 MHz, d6-DMSO) 161.3, 156.4, 146.2, 144.1, 138.9,138.8, 125.8, 125.3, 120.5, 118.9, 115.7, 108.6, 108.4, 104.1, 49.4,29.5, 27.9, 25.6, 24.2, 17.5, ESI-MS m/z: 483 (M + Na)⁺, 461 (M + H)⁺ 59

488 1.78 Activator ¹H NMR (500 MHz, d6- DMSO) δ 11.97 (s, 1H), 10.85 (s,1H), 8.52 (s, 1H), 7.83 (s, 4H), 7.47 (d, J = 3.3 Hz, 1H), 7.38 (d, J =3.3 Hz, 1H), 6.89 (s, 1H), 4.39 (t, J = 7.1 Hz, 2H), 2.66 (s, 6H), 2.20(brs, 2H), 1.87 (t, J = 6.7 Hz, 2H), 1.49 (m, 2H), 1.36-1.19 (m, 8H).¹³C NMR: (125 MHz, d6-DMSO) 161.3, 156.4, 146.2, 144.1, 138.9, 138.8,125.8, 125.4, 120.5, 118.9, 115.8, 108.7, 108.5, 104.1, 49.4, 29.6,28.5, 28.4, 28.2, 25.8, 24.4, 24.3, 17.6, ESI-MS m/z: 487 (M − H)⁻ 60

529 0.63 Activator ¹H NMR (500 MHz, CDCl₃) δ 10.83 (s, 1H), 7.84 (d, J =8.7 Hz, 2H), 7.80 (d, J = 8.8 Hz, 2H), 7.79 (s, 1H), 7.56 (d, J = 3.3Hz, 1H), 7.06 (d, J = 3.3 Hz, 1H), 6.52 (s, 1H), 4.44 (t, J = 6.9 Hz,2H), 2.82 (s, 4H), 2.67 (t, J = 7.0 Hz, 2H), 2.63 (s, 3H), 2.57 (s, 3H),2.14-2.06 (m, 2H), 1.86-1.78 (m, 2H). ¹³C NMR: (125 MHz, CDCl₃) 169.1,168.1, 162.5, 155.7, 148.1, 142.6, 139.7, 139.5, 126.4, 125.2, 119.7,119.1, 117.3, 108.5, 107.1, 105.9, 49.7, 30.5, 29.1, 25.7, 24.8, 21.7,18.3, ESI-MS m/z: 552 (M + Na)⁺, 530 (M + H)⁺ 61

557 0.40 ¹H NMR (500 MHz, CDCl₃) δ 10.84 (s, 1H), 7.85 (d, J = 8.6 Hz,2H), 7.82 (d, J = 8.6 Hz, 2H), 7.75 (s, 1H), 7.57 (d, J = 3.2 Hz, 1H),7.07 (d, J = 3.2 Hz, 1H), 6.53 (s, 1H), 4.40 (t, J = 7.0 Hz, 2H), 2.82(s, 4H), 2.64 (s, 3H), 2.61 (t, J = 7.2 Hz, 2H), 2.58 (s, 3H), 2.02-1.92(m, 2H), 1.81-1.70 (m, 2H), 1.54-1.45 (m, 2H), 1.45-1.36 (m, 2H). ¹³CNMR: (125 MHz, CDCl₃) 169.2, 168.6, 162.5, 155.7, 147.8, 142.6, 139.7,139.5, 126.4, 125.4, 119.6, 119.2, 117.3, 108.5, 107.1, 105.9, 50.2,30.9, 30.1, 28.0, 25.9, 25.7, 24.8, 24.4, 18.3, ESI-MS m/z: 580 (M +Na)⁺, 558 (M + H)⁺ 62

585 0.45 Activator ¹H NMR (500 MHz, CDCl₃) δ 10.84 (s, 1H), 7.85 (d, J =8.7 Hz, 2H), 7.82 (d, J = 8.7 Hz, 2H), 7.73 (s, 1H), 7.57 (d, J = 3.2Hz, 1H), 7.07 (d, J = 3.2 Hz, 1H), 6.53 (s, 1H), 4.39 (t, J = 7.2 Hz,2H), 2.82 (s, 4H), 2.64 (s, 3H), 2.59 (t, J = 7.3 Hz, 2H), 2.58 (s, 3H),2.01-1.89 (m, 2H), 1.81-1.68 (m, 2H), 1.50-1.21 (m, 8H). ¹³C NMR: (125MHz, CDCl₃) 169.3, 168.7, 162.5, 155.7, 147.8, 142.6, 139.7, 139.5,126.4, 125.4, 119.6, 119.0, 117.3, 108.4, 107.1, 105.8, 50.5, 31.0,30.4, 28.8, 28.8, 28.6, 26.4, 25.7, 24.8, 24.6, 18.3, ESI-MS m/z: 608(M + Na)⁺, 586 (M + H)⁺ 63

403 39.81 ¹H NMR (500 MHz, CDCl₃) δ 10.83 (s, 1H), 7.88 (s, 1H), 7.84(d, J = 8.9 Hz, 2H), 7.81 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 3.3 Hz, 1H),7.06 (d, J = 3.3 Hz, 1H), 6.52 (s, 1H), 4.49 (t, J = 6.3 Hz, 2H), 2.82(t, J = 6.3 Hz, 2H), 2.64 (s, 3H), 2.57 (s, 3H), 2.32 (s, 6H) ¹³C NMR:(125 MHz, CDCl₃) 162.5, 155.7, 147.8, 142.6, 139.6, 139.5, 126.4, 125.5,119.8, 119.6, 117.2, 108.4, 107.1, 105.8, 58.9, 48.3, 45.5, 24.8, 18.3,ES-MS m/z: 404 (M + H)⁺ 64

389 7.07 ¹H NMR (500 MHz, d6- DMSO) δ 10.86 (s, 1H), 8.60 (s, 1H), 8.14(brs, 2H), 7.84 (s, 4H), 7.48 (d, J = 3.2 Hz, 1H), 7.37 (d, J = 3.2 Hz,1H), 6.90 (s, 1H), 4.55 (t, J = 6.7 Hz, 2H), 2.85 (s, 2H), 2.65 (s, 6H),2.31-2.11 (m, 2H). ¹³C NMR (125 MHz, d6-DMSO) 161.4, 156.5, 146.3,144.1, 139.1, 138.9, 125.8, 125.2, 120.9, 118.9, 115.8, 108.7, 108.5,104.0, 46.7, 36.2, 27.7, 24.3, 17.6, ESI-MS m/z: 390 (M + H)⁺ 65

419 7.08 ¹H NMR (500 MHz, CDCl₃) δ 10.84 (s, 1H), 7.90 (s, 1H), 7.85 (d,J = 8.7 Hz, 2H), 7.82 (d, J = 8.6 Hz, 1H), 7.57 (t, J = 3.4 Hz, 1H),7.07 (d, J = 3.1 Hz, 1H), 6.53 (s, 1H), 4.59 (t, J = 5.1 Hz, 2H), 3.89(t, J = 5.1 Hz, 2H), 3.55-3.48 (t, J = 4.8 Hz, 2H), 2.95-2.83 (m, 2H),2.64 (s, 3H), 2.58 (s, 3H). ¹³C NMR: (125 MHz, CDCl₃) 162.5, 155.6,147.9, 142.6, 139.7, 139.5, 126.4, 125.4, 120.2, 119.7, 117.3, 108.4,107.1, 105.8, 73.3, 69.4, 50.4, 41.6, 24.8, 18.3 ESI-MS m/z: 420 (M +H)⁺ 66

507 7.94 ¹H NMR (500 MHz, CDCl₃) δ 10.83 (s, 1H), 7.95 (s, 1H), 7.84 (d,J = 8.8 Hz, 2H), 7.81 (d, J = 8.7 Hz, 2H), 7.57 (d, J = 3.3 Hz, 1H),7.07 (d, J = 3.2 Hz, 1H), 6.52 (s, 1H), 4.58 (t, J = 5.0 Hz, 2H), 3.90(t, J = 5.0 Hz, 2H), 3.62 (s, 4H), 3.61-3.54 (m, 4H), 3.45 (t, J = 5.2Hz, 2H), 2.82 (s, 2H), 2.64 (s, 3H), 2.58 (s, 3H). ¹³C NMR: (125 MHz,CDCl₃) 162.5, 155.6, 147.8, 142.6, 139.6, 139.5, 126.4, 125.5, 120.5,119.7, 117.4, 108.4, 107.1, 105.9, 73.3, 70.8, 70.7, 70.6, 70.4, 69.7,50.4, 41.8, 24.8, 18.3, ESI-MS m/z: 508 (M + H)⁺ 67

604 8.91 ¹H NMR (500 MHz, CDCl₃) δ 10.80 (s, 1H), 7.95 (s, 1H), 7.80 (s,4H), 7.51 (d, J = 3.2 Hz, 1H), 7.03 (d, J = 3.2 Hz, 1H), 6.54 (brs, 1H),6.49 (s, 1H), 4.56 (t, J = 4.9 Hz, 2H), 3.89 (t, J = 4.9 Hz, 2H),3.75-3.64 (m, 6H), 3.60 (m, 4H), 3.33 (q, J = 5.7 Hz, 2H), 2.61 (s, 3H),2.53 (s, 3H), 2.47 (d, J = 4.7 Hz, 6H), 2.41 (t, J = 6.0 Hz, 2H). ¹³CNMR: (125 MHz, CDCl₃) 171.1, 162.4, 155.7, 147.7, 142.7, 139.6, 139.5,126.3, 125.4, 120.5, 119.6, 117.0, 108.4, 107.1, 105.5, 70.5, 70.3,69.5, 67.3, 66.6, 57.2, 53.3, 50.3, 36.9, 35.6, 24.8, 18.2, ESI-MS m/z:605 (M + H)⁺ 68

602 10 ¹H NMR (500 MHz, CDCl₃) δ 10.82 (s, 1H), 7.93 (s, 1H), 7.82 (d, J= 8.6 Hz, 2H), 7.79 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 3.0 Hz, 1H), 7.04(d, J = 3.0 Hz, 1H), 6.72 (s, 1H), 6.50 (s, 1H), 4.61-4.46 (m, 2H),3.96-3.79 (m, 2H), 3.67 (t, J = 6.0 Hz, 2H), 3.58 (d, J = 5.5 Hz, 4H),3.38-3.25 (m, 1H), 3.24-3.11 (m, 1H), 3.09- 2.95 (m, 1H), 2.62 (s, 3H),2.55 (s, 3H), 2.36 (t, J = 6.0 Hz, 2H), 2.26 (s, 3H), 2.21-2.06 (m, 2H),1.96-1.83 (m, 1H), 1.81- 1.59 (m, 3H), 1.55- 1.45 (m, J = 13.3, 7.3 Hz,2H). ¹³C NMR: (125 MHz, CDCl₃) 170.9, 162.5, 155.7, 147.8, 142.7, 139.6,139.5, 126.4, 125.4, 120.5, 119.7, 117.2, 108.5, 107.1, 105.7, 70.5,70.3, 69.6, 67.4, 64.7, 57.1, 50.3, 40.5, 37.1, 36.9, 32.0, 29.9, 24.8,22.2, 18.2, ESI-MS m/z: 603 (M + H)⁺ 69

573 2.00 ¹H NMR (500 MHz, CDCl₃) δ 10.83 (s, 1H), 7.93 (s, 1H), 7.85 (d,J = 8.8 Hz, 2H), 7.81 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 3.2 Hz, 1H),7.06 (d, J = 3.3 Hz, 1H), 6.51 (s, 1H), 5.94 (d, J = 7.1 Hz, 1H), 4.57(t, J = 5.0 Hz, 2H), 3.91 (t, J = 5.0 Hz, 2H), 3.77-3.70 (m, 1H), 3.68(t, J = 6.0 Hz, 2H), 3.63- 3.54 (m, 4H), 2.63 (s, 3H), 2.57 (s, 3H),2.36 (t, J = 6.0 Hz, 2H), 1.89- 1.81 (m, 2H), 1.68-1.52 (m, 3H),1.36-1.04 (m, 5H). 13C NMR: (125 MHz, CDCl₃) 170.2, 162.4, 155.7, 147.8,142.7, 139.7, 139.5, 126.4, 125.4, 120.5, 119.6, 117.2, 108.4, 107.1,105.7, 70.5, 70.3, 69.6, 67.5, 50.3, 48.1, 37.3, 33.1, 25.6, 24.9, 24.8,18.2, ESI-MS m/z: 596 (M + Na)⁺, 574 (M + H)⁺ 70

545 3.55 ¹H NMR (500 MHz, CDCl₃) δ 10.88 (s, 1H), 7.96 (s, 1H), 7.88 (d,J = 8.7 Hz, 2H), 7.84 (d, J = 8.7 Hz, 2H), 7.60 (d, J = 3.3 Hz, 1H),7.10 (d, J = 3.3 Hz, 1H), 6.55 (s, 1H), 6.19 (d, J = 6.0 Hz, 1H), 4.59(t, J = 5.0 Hz, 2H), 4.40-4.30 (m, 1H), 3.97- 3.89 (m, 2H), 3.68 (t, J =6.1 Hz, 2H), 3.65-3.57 (m, 4H), 2.66 (s, 3H), 2.61 (s, 3H), 2.35 (t, J =6.1 Hz, 2H), 2.32-2.24 (m, 2H), 1.87-1.77 (m, 2H), 1.70-1.61 (m, 2H).¹³C NMR: (125 MHz, CDCl₃) 170.2, 162.5, 155.7, 147.8, 142.7, 139.7,139.5, 126.4, 125.4, 120.6, 119.7, 117.2, 108.5, 107.1, 105.7, 70.5,70.3, 69.6, 67.4, 50.4, 44.6, 37.0, 31.2, 24.8, 18.3, 15.2, ESI-MS m/z:568 (M + Na)⁺, 546 (M + H)⁺

Example 11 An Activator Modified Glucocerebrosidase for EnzymeReplacement Therapy

Introduction

Gaucher's disease (GD) is a rare genetic lysosomal storage disordercaused by the functional deficiency of glucocerebrosidase (acidβ-glucosidase, GCase) that results in multiple organ malfunction(Futerman and van Meer 2004). Heterozygous mutations in GCase is foundas a major risk factor for Parkinson's disease (PD) (Sidransky, Nalls etal. 2009, Sidransky and Lopez 2012, Lin and Farrer 2014, Schapira,Olanow et al. 2014). Accumulation of glucocerebroside, the substrate ofGCase, in neurons that promotes formation of toxic α-synucleinoligomers, which cause PD (Mazzulli, Xu et al. 2011). Enhance of GCaseactivity is thought to be a potential therapeutic strategy forGCase-associated synucleinopathies, including PD(Sardi, Clarke et al.2013, Sybertz and Krainc 2014).

Treatments for GD include enzyme replacement therapy (ERT) or substratereduction therapy by inhibition of glucosylceramide synthase(Bennett andMohan 2013). Receptor-interacting protein kinase-3 (RIP3) had recentlydiscovered as an emerging therapeutic target of GD(Vitner, Salomon etal. 2014). ERT has proved to be safe and effective over 20 years, and areduction in organ volumes, improvement in hematological parameters andamelioration of bone pains have dramatically improved the quality oflife for many patients (Futerman, Sussman et al. 2004, Weinreb,Goldblatt et al. 2013, Souza, Muniz et al. 2014). There are threerecombinant enzymes with similar activity available for the treatment ofGD today: imiglucerase, velaglucerase alfa, and taliglucerasealfa(Bennett and Mohan 2013) (Tekoah, Tzaban et al. 2013). However,because of the limited effectiveness of these GCase, the cost of ERTranges from US$100,000 to more than $250,000 per year (Grabowski 2008),which impose burdens on patients. Engineering a more-stable enzyme, oran enzyme with a higher catalytic activity, could reduce the number ofinfusions and potentially also reduce cost (Futerman, Sussman et al.2004).

Several different scaffolds of non-iminosugar inhibitory modulators(Zheng, Padia et al. 2007) and non-inhibitory modulator 1 (FIG. 1)(Patnaik, Zheng et al. 2012) were reported since 2007. These modulatorsare suggested to bind in other site than the active site, and stabilizethe enzyme (Zheng, Padia et al. 2007) (Patnaik, Zheng et al. 2012).Modulator 1 can activate purified wt. GCase with AC50 of 5.2 μM andaround 100% maximum activation activity (Patnaik, Zheng et al. 2012). Toimprove the GCase enzyme activity and stability for ERT and study theactivation mechanism of the modulators, in current study, we firstlyreport the high activation activators and an activator covalent modifiedGCase.

Results

Discovery of High Activation Activators. To develop potent and highactivation activators, the pyrazolopyrimidine ring of 1 was modified togive pyrrolopyrimidine scaffold compounds exhibiting higher enzymeactivity. The alkyne group on the phenyl ring was coupled with an azidebearing a polyethylene glycol (PEG₂) linker by click reaction to give 2(Scheme 1). With around 14 folds of maximum activity and AC₅₀ of 6.31 μMin 4MU-Glc substrate enzyme activity assay (FIG. 2A), 2 is, to ourknowledge, among the most potent activators yet identified. Moreover, 2highly activated (10 folds) Res-β-glc substrate hydrolysis at 50 μM,while lead compound 1 did not show any activation activity in theRes-β-glc substrate enzyme activity assay. Consistent with the previousreport (Patnaik, Zheng et al. 2012), none of these compoundsdemonstrated any activation activity in current in vitro naturalsubstrate assay.

Design of the Fluorescent Probe for Fluorescence Polarization Assay. Tofacilitate the detection of the activator binding affinity, compound 2was coupling with a fluorophore, to afford the probe 3 (Scheme 1). Thefluorescence polarization (FP) signal was gradually saturated with theincreased concentration of the enzyme (FIG. 3A). Probe 3 was observed tohave a potent binding affinity with a Kd value of 0.71 μM, indicatingthat the probe 3 could bind to GCase directly. To determine the bindingaffinity of the activators, probe 3 was competed with differentconcentrations of compound 1 and 2, respectively. Compound 1 showed veryweak activity in this assay, while compound 2 exhibited much higheractivity (FIG. 3B).

Design of an Electrophilic Probe 4 and the Covalent Binding Assay. Probe4 was designed and synthesized to target the lysine residues surroundingthe binding site by the reaction of NHS ester with primary amine (Scheme1). The probe 4 also showed high activation activity in both of the4MU-Glc (20 folds) and red substrates (10 folds) enzyme activity assays(FIGS. 2A and 2B).

The probe 4 was subjected to the covalent binding assay against GCase.The reaction conditions, including the buffer pH value, theconcentration of the protein and probe, and the reaction temperaturewere carefully adjusted to achieve the maximum activation. With theoptimized reaction condition, the enzyme activity was activated in atime-dependent manner (FIG. 5). The enzyme could be activated by probe 4with around 22 and 28 folds activity comparing to the wt. GCase in the4MU-Glc substrate (FIG. 5A) and red substrate (FIG. 5B) enzyme activityassay respectively. To our surprise, the covalent activated enzyme alsodemonstrated 16 folds activity improvement in the natural substrateassay (FIG. 5C). The enzyme activity was not decreased significantlyfollowed by dialysis in three substrate assays, indicating that theenzyme had been covalent modified by probe 4. This result was alsoconfirmed by high-resolution LC-MS spectrum, indicating that GCase wasmodified by 1-3 ligands, despite the presence of twenty two lysineresidues on the protein.

Saposin C Compete Assay with Probe 4 Pre-activated GCase. The wt. GCasecould be activated by Saposin C(Tamargo, Velayati et al. 2012), anatural co-factor of GCase in a dose-dependent manner with an AC₅₀ valueof 100 nM (FIG. 5). However, the binding site and binding mode ofsaposin C remains unknown. To investigate whether saposin C and probe 4share the same binding site, the covalent activated GCase was titratedwith saposin C, and the enzyme activity was tested in the 4MU-Glcsubstrate enzyme activity assay. The pre-activated enzyme could befurther activated by saposin C in a dose-dependent manner with an AC₅₀value of 71 nM (FIG. 5). It seemed that the enzyme activity wasmodulated by the activator and saposin C in a synergy mode, suggestingthat the binding site of the probe 4 and saposin C are different.

Enzyme Stability Assay in pH 4.7 Buffer and Human Plasma. To testwhether probe 4 changed the enzyme stability of GCase, we used twoenvironments for GCase activity stability assay (Tekoah, Tzaban et al.2013). The first one was citrate/phosphorylate buffer, which was at pH4.7. In this condition, the wt. GCase only had about 15% activity lefton day 4 (FIGS. 6A and 6B), which is similar to the previous research(Tekoah, Tzaban et al. 2013). An interesting finding was that even onday 10, activated GCase still had more than 20% activity left. Thisresult suggested that the activated GCase not only had higher absoluteGCase activity on each day, but also showed a strong delay of activityreduction after normalization in low pH buffer. One possible reason forthe higher enzyme activity in low pH buffer was that activated GCase wasstabilized by the covalent compound, and was more difficult to unfold.

Then we tested both the activated GCase and wt. GCase in human plasma(pH 7.4). To our surprise, even though the activated GCase had higheractivity at each time point, after normalization, the activated GCaseand wt. GCase showed similar enzyme activity stability curves (FIGS. 6Cand 6D). This result suggested that probe 4 didn't affect thedegradation of GCase in human plasma.

U937 Macrophage Uptake Assay. The following question we asked waswhether human macrophage cell, GD's target cell, could uptake activatedGCase normally. After PMA induction, we treated the U937 cell line withboth activated GCase and wt. GCase (Tekoah, Tzaban et al. 2013). Theactivated GCase showed more than 25 folds activity compared toendogenous GC, while wt. GCase had only about 3 folds activity (FIG.7A), despite the protein uptake level of the activated GCase and wt.GCase were equal (FIG. 7B). These results indicated that probe 4 wouldnot affect the uptake of GCase into macrophage cells but it couldimprove GCase enzyme activity in cells.

Discussion

In over two decades, enzyme replacement therapy (ERT) had achieved greatsuccess in the treatment of genetic lysosomal storage diseases, such asGaucher's disease (Futerman, Sussman et al. 2004) Fabry disease(Pisani,Visciano et al. 2012) and Pompe disease(Angelini and Semplicini 2012).However, the major issue of ERT is the cost of the enzyme treatment(Grabowski 2008), which preventing many of the patients access thistherapy. In this study, we discovered the high activation compounds anduse them for specific modification of recombinant glucocerebrosidase toimprove the enzyme activity as well as stability.

Lead compound 1 was discovered as a non-inhibitory chaperone in 2012(Patnaik, Zheng et al. 2012). In our effort to discover potentactivators, according to our SAR study, a new pyrrolopyrimidine scaffoldhad been found to have high activation activity and binding affinitythan 1. Because of low solubility of these activators in aqueoussolution, further modification on the para position of the phenyl ringwith a trizole ring bearing a PEG linker had been discovered to presenthigher activation activity and better solubility. Compound 2, with aPEG₂ linker, was found to give the highest activation in these series ofactivators.

To evaluate the SAR of these activators, a binding assay is needed toevaluate their binding affinity. The only detection method reported forthe binding of GCase and the activators is microscale thermophoresis(MST) (Patnaik, Zheng et al. 2012). Some other biophysics techniques,such as isothermal titration calorimetry (ITC), surface plasmonresonance (SPR), and fluorescence thermal shift, had been attempted tofind out the binding of the GCase activators. Unfortunately, thesemethods failed to give any positive result in our study. Fluorescencepolarization (FP) assay is a homogeneous method that allows rapid andquantitative analysis of diverse molecular interactions and enzymeactivities(Rossi and Taylor 2011). A fluorescent probe was designed andsynthesized to measure the binding affinity of the probe and GCase.Probe 3 was measured to have an Kd value around 0.71 μM, indicating thatthis probe binds tightly with GCase. With this FP assay, we could detectthe binding of the compounds with GCase, whatever they are activators,inhibitors or just binders. So this method could be further utilized inthe high throughput screening (HTS) to discover more diverse compoundsas the modulators of GCase.

Since we have the high activation activators in hand, we hypothesizedthat once GCase was covalent modified by our compound, the enzymeactivity could be enhanced to the maximum activity of the activatorbecause the local concentration of the activator is high. Primary amineof lysine residue, a high reactive group exists on most protein surface,could be modified by many diverse chemicals(Nakamura, Kawai et al. 2009,Choi, Connelly et al. 2010). NHS ester had been use to react with lysinein a plenty of biological studies(Nanda and Lorsch 2014). Based on ourSAR study, we found a PEG2 linker was the suitable length to achievemaximum enzyme activity. In the covalent binding assay, wt. GCase wasmodified and activated by probe 4 in a time-dependent manner. Themaximum enzyme activity could be achieved by using two equivalent of 4in one hour at pH 7.2. Either higher pH value or increased ratio ofprobe 4 could reduce the enzyme activity, suggesting that thenon-specified binding of probe 4 will affect the enzyme activity.Considering of the conditions we used in the covalent binding assay,this result demonstrated that the elevated enzyme activity coming fromthe scaffold of the ligand and the specific covalent modification of theenzyme.

Saposin C binding mode had been studied and modeled(Atrian, Lopez-Vinaset al. 2008). If the activated enzyme could not be activated by SaposinC, the binding site of the activator and Saposin C would be the same.Actually, the activated GCase could be further activated by Saposin C.The modification of the activator did not interfere with Saposin Cinteraction, indicating that the binding site of the activator isdifferent from Saposin C. The result also suggested that our highactivated GCase may not require Saposin C for further activation. Thisenzyme could be used for the patients with Saposin C mutation.

To evaluate whether the activated enzyme could be used in ERT, weexamined the enzyme stability and cell uptake property. The activatedenzyme demonstrated much more stability in acidic buffer and samestability in human plasma to wt. GCase. In U937 uptake assay, the sameamount of the activated GCase could be uptaken by PMA induced U937macrophage cell comparing to wt. GCase, indicating that compoundmodification does not affect the GCase uptake process. With much higherenzyme activity and stability of the activated GCase in the acidiccondition, as demonstrated in this study, we may suggest that diminishedinjection dose of this enzyme might be used in ERT for GD.

In addition, more different recombinant lysosomal enzymes, such asα-1-iduronidase, α-galactosidase, α-glucosidase, N-acetylgalactosamine4-sulfatase and iduronate sulfatase, had been approved to treat fortheir respective deficiency diseases (MPS I, Fabry disease, Pompedisease, MPS VI and MPS II)(Desnick and Schuchman 2012, Valayannopoulos2013). Wide application of our activator modified enzyme approach hasthe potential to improve the treatment in other lysosomal storagediseases.

Methods

Biology

4-Methylumbelliferyl β-D-glucopyranoside (4MU-β-glc), a blue-fluorogenicsubstrate, resorufin β-D-glucopyranoside (Res-β-glc), a red-fluorogenicsubstrate, and the buffer components were purchased from Sigma-Aldrich(St. Louis, Mo.). Natural substrate, glucosylceramide was purchased fromAvanti Polar Lipids, Inc. (Alabaster, Alabama). The Amplex RedGlucose/Glucose Oxidase Assay Kit was purchased from Invitrogen (Eugene,Oreg.) to measure the amount of glucose produced when glucosylceramideis cleaved by glucocerebrosidase.

The recombinant wt. enzyme velaglucerase alfa (Vpriv®, Shire HumanGenetic Therapies, Inc.) was obtained from residual solution afterclinical infusions. The GCase activity assay buffer was composed of 50mM citric acid, 176 mM K₂HPO₄, and 0.01% Tween-20 at pH 5.9. A solutionof 1 M sodium hydroxide and 1 M glycine was used as the stop solutionfor the 4MU-β-glc substrate assay.

Compound Activity Assay with 4MU-β-glc Substrate and Res-R-glcSubstrate. The compounds in DMSO solution 0.5 μL/well was transferred toa black 96-well plate (the final titration was 24 nM to 50 μM, 12concentrations). 33.5 μL enzyme solution (7.5 nM final concentration)was transferred to the wells. After 5 min of incubation at roomtemperature, the enzyme reaction was initiated by the addition of 33μL/well 4MU-Glc substrate or 66.5 μL/well red substrate. Finalconcentrations of the 4MU-Glc substrate and Res-Glc substrate were 1.5mM and 30 μM, respectively. The red substrate reaction was measured inthe Biotek Synergy H1 multi-mode plate reader with Ex=573 nm and Em=610nm at 37° C. in every 20 seconds for 30 min. The 4MU-Glc substratereaction was terminated by the addition of 33 μL/well stop solution (1 MNaOH and 1 M glycine mixture, pH 10) after 30 min of incubation at 37°C. The fluorescence was then measured in the plate reader with Ex=365 nmand Em=440 nm.

Compound Activity Assay with Natural Substrate. The compounds weretested with natural substrate by using the slight modified method asdescribed previously (Motabar, Goldin et al. 2012). The compounds inDMSO solution 0.5 μL/well was transferred to a black 96-well plate (thefinal titration was 24 nM to 50 KM, 12 concentrations). 33.5 μL enzymesolution was transferred to the wells. After 5 min of incubation at roomtemperature, the enzyme reaction was initiated by the addition of 16μL/well natural substrate. Final concentrations of the natural substrate(glucosylceramide) was 100 μM. The plate was incubated for 30 min at 37°C., and was added the Amplex Red Glucose/Glucose Oxidase Assay buffer(50 μL/well). The plate was measured in the Biotek Synergy H1 multi-modeplate reader with Ex=573 nm and Em=610 nm at 37° C. every 20 seconds for30 min.

Fluorescence Polarization (FP) Assay. The fluorescent probe 3 (25nL/well, 50 nM final concentration) was transferred to a 384-wells blackplate by using Labcyte Echo 550 Liquid Handler system. The 25 μL/wellenzyme dilutions in GCase enzyme activity buffer (the final titrationwas 5 nM to 10 μM, 10 concentrations, 2 times dilution) were added tothe plate, and shook at room temperature in dark for 20 min. Thefluorescence polarization was measured in Molecular Devices Analyst GTwith Ex=535 nm and Em=580 nm, G Factor=1.05.

Binding Affinity Test by FP Assay. The enzyme in GCase enzyme activitybuffer (25 μL/well) was added to a 384-wells black plate, Thefluorescent probe 3 (25 nL/well, 50 nM final concentration) wastransferred to a 384-wells black plate by using Labcyte Echo 550 LiquidHandler system. Compounds 1, and 2 in DMSO stock solution (50 nL) (thefinal titration was 19.5 nM to 10 μM, 10 concentrations) weretransferred to the plate. The plate was shook at room temperature indark for 20 min. The fluorescence polarization was measured in MolecularDevices Analyst GT with Ex=535 nm and Em=580 nm, G Factor=1.05.

Saposin C Activation Assay with 4MU-Glc Substrate. Saposin C (0.5μL/well) was added to a black 96-well plate (the final titration was1.72 nM to 1.76 μM, 12 concentrations). The compound 4 activated enzymeand wt. enzyme (33.5 μL/well, 7.5 nM final concentration) in assaybuffer (50 mM Citric acid, 176 mM K₂HPO₄, 0.01% tween-20, 0.01% (g/mL)phosphatidylserine, pH 4.7) was added to the wells, respectively. After5 min of incubation at room temperature, the enzyme reaction wasinitiated by the addition of 33 μL/well 4MU-Glc substrate. Finalconcentrations of the 4MU-Glc substrate was 1.5 mM. The reaction wasterminated by the addition of 33 μL/well stop solution (1 M NaOH and 1 Mglycine mixture, pH 10) after 30 min of incubation at 37° C. Thefluorescence was then measured in the plate reader with Ex=365 nm andEm=440 nm.

High Resolution HPLC-MS Spectrum. The enzyme was treated with compound 4at pH 7.2 for 60 min as described above. The sample was dialyzed with0.1 M Tris pH 7.2 buffer and analyzed on Agilent 6210A LC-TOF massspectrometer equipped with a C8 column.

Differentiation of Human U937 Macrophage Cells. Human monocyte cell lineU937 was optimally differentiated into macrophages by the addition of 75ng/ml PMA (Brumshtein, Salinas et al. 2010) to the monocyte culture for3 days (in 75 cm² flasks). Macrophages were enriched by adhesion to theculture plate and identified by morphology and receptor staining.

GCase Uptake Studies in Macrophage Cells. The assay was carried outaccording to the reported method (Tekoah, Tzaban et al. 2013) withslight modification. The enzyme was treated with compound 4 or DMSO atpH 7.2 for 60 min as described above, then dialyzed with PBS. U937cells, after differentiation (˜1.6*106 cells), were incubated with 60μg/ml of the activated enzymes and wt. enzyme in F12K medium for 10 minat 5% CO₂, 3TC incubator. The cells were then washed twice with ice coldPBS enriched with Mannan (1 mg/ml). To further ensure release of anyproteins bound non-specifically to the membrane half of the cells werewashed with ice cold glycine buffer: (0.8% NaCl, 0.038% KCl, 0.01%MgCl2, 0.01% CaCl₂, 0.7% glycine [pH 3]) followed by two additional coldPBS washes. The other half of the wells were used as controls formeasuring total enzymatic activity (bound and internalized enzyme). Thecells were lysed by adding β-glucocerebrosidase activity buffer (60 mMphosphate-citrate buffer; 0.15% Triton X-100; 0.125% sodiumtaurocholate, pH 5.5), followed by pipetting and one freeze/thaw cycle.The obtained lysates were subjected to determination of enzymaticactivity using the colorimetric method detailed above. The totalenzymatic activity was normalized to total soluble proteins as measuredby Bradford assay. The effective uptake was calculated as the percentageof activity within the cells out of the total enzymatic activity of thecontrols.

Chemistry

Commercially available reagents and solvents were used without furtherpurification. Compounds were synthesized and analyzed as indicated inthis example and the foregoing examples. All reactions were monitored bythin layer chromatography (TLC) with 0.25 mm Silicycle extra hard 250 μMTLC plates (60 F254). Purification of reaction products was carried outby flash chromatography using Agilent 971-FP flash purification systemwith Silicycle Silica Gel columns (4 g, 12 g, 24 g, 40 g or 80g). Thepurity of all compounds was over 95% and was analyzed with Agilent 1260Infinity HPLC system. NMR spectra and ¹³C NMR were obtained using aBruker Avance III 500 MHz system (500 MHz for ¹H NMR and 125 MHz for ¹³CNMR) spectrometer. Chemical shifts are reported relative to chloroform(8=7.26) for NMR and chloroform (8=77.16) for ¹³C NMR or dimethylsulfoxide (8=2.50) for and dimethyl sulfoxide (8=39.52) for ¹³C NMR.Data are reported as (br=broad, s=singlet, d=doublet, t=triplet,q=quartet, m=multiplet). Mass spectra were obtained using Bruker AmaZonSL system.

REFERENCES

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Atrian, S., et al. (2008). “An evolutionary and structure-based dockingmodel for glucocerebrosidase-saposin C and glucocerebrosidase-substrateinteractions—relevance for Gaucher disease.” Proteins 70(3): 882-891.

Bennett, L. L. and D. Mohan (2013). “Gaucher disease and its treatmentoptions.” Ann Pharmacother 47(9): 1182-1193.

Brumshtein, B., et al. (2010). “Characterization of gene-activated humanacid-beta-glucosidase: crystal structure, glycan composition, andinternalization into macrophages.” Glycobiology 20(1): 24-32.

Choi, S., et al. (2010). “Chemoselective small molecules that covalentlymodify one lysine in a non-enzyme protein in plasma.” Nature ChemicalBiology 6(2): 133-139.

Desnick, R. J. and E. H. Schuchman (2012). “Enzyme replacement therapyfor lysosomal diseases: lessons from 20 years of experience andremaining challenges.” Annu Rev Genomics Hum Genet 13: 307-335.

Futerman, A. H., et al. (2004). “New directions in the treatment ofGaucher disease.” Trends in Pharmacological Sciences 25(3): 147-151.

Futerman, A. H. and G. van Meer (2004). “The cell biology of lysosomalstorage disorders.” Nat Rev Mol Cell Biol 5(7): 554-565.

Grabowski, G. A. (2008). “Phenotype, diagnosis, and treatment ofGaucher's disease.” Lancet 372(9645): 1263-1271.

Lin, M. K. and M. J. Farrer (2014). “Genetics and genomics ofParkinson's disease.” Genome Medicine 6.

Mazzulli, J. R., et al. (2011). “Gaucher disease glucocerebrosidase andalpha-synuclein form a bidirectional pathogenic loop insynucleinopathies.” Cell 146(1): 37-52.

Motabar, O., et al. (2012). “A high throughput glucocerebrosidase assayusing the natural substrate glucosylceramide.” Anal Bioanal Chem 402(2):731-739.

Nakamura, T., et al. (2009). “Covalent modification of lysine residuesby allyl isothiocyanate in physiological conditions: plausibletransformation of isothiocyanate from thiol to amine.” Chem Res Toxicol22(3): 536-542.

Nanda, J. S. and J. R. Lorsch (2014). “Labeling a Protein withFluorophores Using NHS Ester Derivitization.” Laboratory Methods inEnzymology: Protein Pt A 536: 87-94.

Patnaik, S., et al. (2012). “Discovery, structure-activity relationship,and biological evaluation of noninhibitory small molecule chaperones ofglucocerebrosidase.” J Med Chem 55(12): 5734-5748.

Pisani, A., et al. (2012). “Enzyme replacement therapy in patients withFabry disease: state of the art and review of the literature.” Mol GenetMetab 107(3): 267-275.

Rossi, A. M. and C. W. Taylor (2011). “Analysis of protein-ligandinteractions by fluorescence polarization.” Nat Protoc 6(3): 365-387.

Sardi, S. P., et al. (2013). “Augmenting CNS glucocerebrosidase activityas a therapeutic strategy for parkinsonism and other Gaucher-relatedsynucleinopathies.” Proceedings of the National Academy of Sciences ofthe United States of America 110(9): 3537-3542.

Schapira, A. H. V., et al. (2014). “Slowing of neurodegeneration inParkinson's disease and Huntington's disease: future therapeuticperspectives.” Lancet 384(9942): 545-555.

Sidransky, E. and G. Lopez (2012). “The link between the GBA gene andparkinsonism.” Lancet Neurology 11(11): 986-998.

Sidransky, E., et al. (2009). “Multicenter analysis ofglucocerebrosidase mutations in Parkinson's disease.” N Engl J Med361(17): 1651-1661.

Souza, A. M., et al. (2014). “Study of enzyme replacement therapy forGaucher Disease: comparative analysis of clinical and laboratoryparameters at diagnosis and after two, five and ten years of treatment.”Rev Bras Hematol Hemoter 36(5): 345-350.

Sybertz, E. and D. Krainc (2014). “Development of targeted therapies forParkinson's disease and related synucleinopathies.” J Lipid Res 55(10):1996-2003.

Tamargo, R. J., et al. (2012). “The role of saposin C in Gaucherdisease.” Mol Genet Metab 106(3): 257-263.

Tekoah, Y., et al. (2013). “Glycosylation and functionality ofrecombinant beta-glucocerebrosidase from various production systems.”Bioscience Reports 33: 771-U272.

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Example 12 Synthesis and Testing of Additional Substituted OuinazolineCompounds

Additional compounds were prepared and tested according to theprocedures provided in the examples above. Results are shown in Table 2.

TABLE 2 Activity AC₅₀ or IC₅₀ No. Structure MW (μM) 71

1140  1.26  activator 72

370  6.13  activator 73

391  0.316 activator 74

459  0.71  activator 75

488  1.26  activator 76

660 28.18  activator 77

537 NA 78

522  0.71  activator 79

508  3.54  activator 80

389 activator 81

393  1.78  activator 82

542  0.8   activator 83

605  0.56  activator 84

85

86

87

88

89

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein. The terms and expressions whichhave been employed are used as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention. Thus, itshould be understood that although the present invention has beenillustrated by specific embodiments and optional features, modificationand/or variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

We claim:
 1. A compound or a salt or solvate thereof having a Formula I:

wherein: R¹ is hydrogen; a C1-C6 alkyl group; a C2-C6 alkenyl group; aC2-C6 alkynyl group; a saturated or unsaturated homocycle or heterocyclecomprising one 5- or 6-membered ring, a saturated or unsaturatedhomocycle or heterocycle comprising two fused 5- or 6-membered rings, oran alkylthiophene; and R¹ optionally is substituted at one or morepositions with a C1-C6 alkyl group, a C1-C6 alkoxy group, a halo group,a haloalkyl group, phenyl group which optionally is substituted withhalo, a benzyl group which optionally is substituted with halo, atriazole group optionally substituted with a carboxyl group, a2,5-dioxopyrrolidinyl-1-yl-carboxylate group, an amino group, analkyl-N,N-dialkyl amino group, an alkyl-alkyoxy-amino group, analkyl-alkyoxy-alkoxy-amino group, analkyl-alkyoxy-alkoxy-carboxamide-alkyl-morpholine group, analkyl-alkyoxy-alkoxy-carboxamide-alkyl-1-alkylpyrrolidine group, analkyl-alkyoxy-alkoxy-carboxamide-alkyl-cyclohexyl group, or analkyl-alkyoxy-alkoxy-carboxamide-alkyl-cyclobutyl group; an imidazolegroup; a pyridyl group optionally substituted with phenoxy; apyrrolidinyl group, a piperazinyl group, a 4-alkylpiperazine group, a4-benzylpiperazine group, an alkyl-4-alkylpiperazine group; apiperidinyl group; a 4-alkylpiperidine group; a4-N,N,diakylaminopiperidine group; a morpholinyl group; analkylmorpholine group; an amino group; an alkylamino group; a dialkylamino group; an alkyl-N,N-dialkylamino group; an azide group; a hydroxylgroup; an alkylhydroxyl group; an alkynylphenyl group; a phenylmethanonegroup; an oxyphenyl group; an oxycarboxyl group, or R¹ has a formulaselected from:

and R³ has a formula selected from

and R⁴ is H, C1-C8 alkyl, phenyl, or succinimidyl; and R² is C1-C6 alkylor pyridinyl (e.g., 2-yl, 3-yl, or 4-yl).
 2. The compound of claim 1,wherein R¹ is selected from the group consisting of


3. The compound of claim 1 having Formula IA:

wherein: R² is hydrogen a C1-C6 alkyl group; a C2-C6 alkenyl group; aC2-C6 alkynyl group; a C1-C6 alkoxy group; a halo group; a haloalkylgroup; a phenyl group; a benzyl group; a triazole group optionallysubstituted with a carboxylic acid group; a2,5-dioxopyrrolidinyl-1-yl-carboxylate group; an amino group; analkyl-N,N-dialkyl amino group; an alkyl-alkyoxy-amino group; analkyl-alkyoxy-alkoxy-amino group; analkyl-alkyoxy-alkoxy-carboxamide-alkyl-morpholine group; analkyl-alkyoxy-alkoxy-carboxamide-alkyl-1-alkylpyrrolidine group; analkyl-alkyoxy-alkoxy-carboxamide-alkyl-cyclohexyl group; and analkyl-alkyoxy-alkoxy-carboxamide-alkyl-cyclobutyl group; an imidazolegroup; a pyrrolidine group; a piperazine group; a 4-alkylpiperazinegroup; a 4-benzylpiperazine group; an alkyl-4-alkylpiperazine group; apiperidine group; a 4-alkylpiperidine group; a4-N,N,diakylaminopiperidine group; a morpholine group; analkylmorpholine group; an amino group; an alkylamino group; a dialkylamino group; an alkyl-N,N-dialkylamino group; an azide group; a hydroxylgroup; an alkylhydroxyl group; an alkynylphenyl group; a phenylmethanonegroup; an oxyphenyl group; or an oxycarboxylic acid group.
 4. Thecompound of claim 1 having Formula IB:

wherein: R³ is a carboxyl group, a2,5-dioxopyrrolidinyl-1-yl-carboxylate group; an alkylamino group; analkyl-N,N-dialkyl amino group; an alkyl-alkyoxy-amino group; analkyl-alkyoxy-alkoxy-amino group; analkyl-alkyoxy-alkoxy-carboxamide-alkyl-morpholine group; analkyl-alkyoxy-alkoxy-carboxamide-alkyl-1-alkylpyrrolidine group; analkyl-alkyoxy-alkoxy-carboxamide-alkyl-cyclohexyl group; and analkyl-alkyoxy-alkoxy-carboxamide-alkyl-cyclobutyl group.
 5. Thecompounds of claim 1 wherein any of R¹, R², and R³ comprises afluorophore.
 6. The compound of claim 1 having Formula ID:

wherein: X is N or S; and R⁴, R⁵, R⁶, and R⁷, are each independentlyselected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, and halogen.
 7. Apharmaceutical composition comprising the compound of claim 1 and apharmaceutical carrier.
 8. A method for treating a disease or disorderthat is associated with glucocerebrosidase activity, the methodcomprising administering the composition of claim
 7. 9. The method ofclaim 8, wherein the disease or disorder is a neurological disease ordisorder.
 10. The method of claim 9, wherein the neurological disease ordisorder is a degenerative neurological disease or disorder.
 11. Themethod of claim 10, wherein the degenerative neurological disease ordisorder is Gaucher's disease.
 12. The method of claim 10, wherein thedegenerative neurological disease or disorder is Parkinson's disease.13. A fluorescent probe comprising the compound of claim
 5. 14. A methodof screening for a compound that binds to glucocerebrosidase, the methodcomprising contacting glucocerebrosidase with the fluorescent probe ofclaim 13 and observing fluorescence polarization.
 15. Activatedglucocerebrosidase comprising glucocerebrosidase covalently attached tothe compound of claim
 1. 16. A pharmaceutical composition comprising theactivated glucocerebrosidase of claim 15 and a pharmaceutical carrier.17. A method for treating a disease or disorder that is associated withglucocerebrosidase activity, the method comprising administering thecomposition of claim
 16. 18. The method of claim 17, wherein the diseaseor disorder is a neurological disease or disorder.
 19. The method ofclaim 18, wherein the disease or disorder is a degenerative neurologicaldisease or disorder.
 20. The method of claim 19, wherein thedegenerative neurological disease or disorder is Gaucher's disease.